Hepatitis C virus inhibitors

ABSTRACT

Hepatitis C virus inhibitors are disclosed having the general formula:  
                 
 
wherein R 1 , R 2 , R 3 , R′, B, Y and X are described in the description. Compositions comprising the compounds and methods for using the compounds to inhibit HCV are also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This non-provisional application claims priority from the provisionalapplication U.S. Ser. No. 60/523,764 filed Nov. 20, 2003.

FIELD OF THE INVENTION

The present invention is generally directed to antiviral compounds, andmore specifically directed to compounds which inhibit the functioning ofthe NS3 protease (also referred to herein as “serine protease”) encodedby Hepatitis C virus (HCV), compositions comprising such compounds andmethods for inhibiting the functioning of the NS3 protease.

BACKGROUND OF THE INVENTION

HCV is a major human pathogen, infecting an estimated 170 millionpersons worldwide—roughly five times the number infected by humanimmunodeficiency virus type 1. A substantial fraction of these HCVinfected individuals develop serious progressive liver disease,including cirrhosis and hepatocellular carcinoma. (Lauer, G. M.; Walker,B. D. N. Engl. J. Med. (2001), 345, 41-52).

Presently, the most effective HCV therapy employs a combination ofalpha-interferon and ribavirin, leading to sustained efficacy in 40% ofpatients. (Poynard, T. et al. Lancet (1998), 352, 1426-1432). Recentclinical results demonstrate that pegylated alpha-interferon is superiorto unmodified alpha-interferon as monotherapy (Zeuzem, S. et al. N.Engl. J. Med. (2000), 343, 1666-1672). However, even with experimentaltherapeutic regimens involving combinations of pegylatedalpha-interferon and ribavirin, a substantial fraction of patients donot have a sustained reduction in viral load. Thus, there is a clear andlong-felt need to develop effective therapeutics for treatment of HCVinfection.

HCV is a positive-stranded RNA virus. Based on a comparison of thededuced amino acid sequence and the extensive similarity in the 5′untranslated region, HCV has been classified as a separate genus in theFlaviviridae family. All members of the Flaviviridae family haveenveloped virions that contain a positive stranded RNA genome encodingall known virus-specific proteins via translation of a single,uninterrupted, open reading frame.

Considerable heterogeneity is found within the nucleotide and encodedamino acid sequence throughout the HCV genome. At least six majorgenotypes have been characterized, and more than 50 subtypes have beendescribed. The major genotypes of HCV differ in their distributionworldwide, and the clinical significance of the genetic heterogeneity ofHCV remains elusive despite numerous studies of the possible effect ofgenotypes on pathogenesis and therapy.

The single strand HCV RNA genome is approximately 9500 nucleotides inlength and has a single open reading frame (ORF) encoding a single largepolyprotein of about 3000 amino acids. In infected cells, thispolyprotein is cleaved at multiple sites by cellular and viral proteasesto produce the structural and non-structural (NS) proteins. In the caseof HCV, the generation of mature non-structural proteins (NS2, NS3,NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. Thefirst one is believed to cleave at the NS2-NS3 junction; the second oneis a serine protease contained within the N-terminal region of NS3 andmediates all the subsequent cleavages downstream of NS3, both in cis, atthe NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B,NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiplefunctions, acting as a cofactor for the NS3 protease and possiblyassisting in the membrane localization of NS3 and other viral replicasecomponents. The complex formation of the NS3 protein with NS4A seemsnecessary to the processing events, enhancing the proteolytic efficiencyat all of the sites. The NS3 protein also exhibits nucleosidetriphosphatase and RNA helicase activities. NS5B is a RNA-dependent RNApolymerase that is involved in the replication of HCV.

Among the compounds that have demonstrated efficacy in inhibiting HCVreplication, as selective HCV serine protease inhibitors, are thepeptide compounds disclosed in U.S. Pat. No. 6,323,180.

SUMMARY OF THE INVENTION

The present invention provides compounds of formula I;

wherein:

-   -   (a) R₁ is Het or aryl;    -   (b) m is 1 or 2;    -   (c) n is 1 or 2;    -   (d) R₂ is H; or C₁₋₆ alkyl, C₂₋₆ alkenyl or C₃₋₇ cycloalkyl,        each optionally substituted with halogen;    -   (e) R₃ is C₁₋₈ alkyl optionally substituted with halo, cyano,        amino, C₁₋₆ dialkylamino, C₆₋₁₀ aryl, C₇₋₁₄ alkylaryl, C₁₋₆        alkoxy, carboxy, hydroxy, aryloxy, C₇₋₁₄ alkylaryloxy, C₂₋₆        alkylester or C₈₋₁₅ alkylarylester; C₃₋₁₂ alkenyl, C₃₋₇        cycloalkyl, or C₄₋₁₀ alkylcycloalkyl, wherein the cycloalkyl or        alkylcycloalkyl are optionally substituted with hydroxy, C₁₋₆        alkyl, C₂₋₆ alkenyl or C₁₋₆ alkoxy; or R₃ together with the        carbon atom to which it is attached forms a C₃₋₇ cycloalkyl        group optionally substituted with C₂₋₆ alkenyl;    -   (f) Y is H, phenyl substituted with nitro, pyridyl substituted        with nitro, or C₁₋₆ alkyl optionally substituted with cyano, OH        or C₃₋₇ cycloalkyl; provided that if R₄ or R₅ is H then Y is H;    -   (g) B is H, C₁₋₆ alkyl, R₄—(C═O)—, R₄O(C═O)—, R₄—N(R₅)—C(═O)—,        R₄—N(R₅)—C(═S)—, R₄SO₂—, or R₄—N(R₅)—SO₂—;    -   (h) R₄ is (i) C₁₋₁₀ alkyl optionally substituted with phenyl,        carboxyl, C₁₋₆ alkanoyl, 1-3 halogen, hydroxy, —OC(O)C₁₋₆ alkyl,        C₁₋₆ alkoxy, amino optionally substituted with C₁₋₆ alkyl,        amido, or (lower alkyl) amido; (ii) C₃₋₇ cycloalkyl, C₃₋₇        cycloalkoxy, or C₄₋₁₀ alkylcycloalklyl, each optionally        substituted with hydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl, amino        optionally substituted with C₁₋₆ alkyl, amido, or (lower alkyl)        amido; (iii) C₆₋₁₀ aryl or C₇₋₁₆ arylalkyl, each optionally        substituted with C₁₋₆ alkyl, halogen, nitro, hydroxy, amido,        (lower alkyl) amido, or amino optionally substituted with C₁₋₆        alkyl; (iv) Het; (v) bicyclo(1.1.1)pentane; or (vi) —C(O)OC₁₋₆        alkyl, C₂₋₆ alkenyl or C₂₋₆ alkynyl;    -   (i) R₅ is H; C₁₋₆ alkyl optionally substituted with 1-3        halogens; or C₁₋₆ alkoxy provided R₄ is C₁₋₁₀ alkyl;    -   (j) X is O, S, SO, SO₂, OCH₂, CH₂O or NH;    -   (k) R′ is Het, C₆₋₁₀ aryl or C₇₋₁₄ alkylaryl, each optionally        substituted with R^(a); and    -   (l) R^(a) is C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₁₋₆ alkoxy, C₃₋₇        cycloalkoxy, halo-C₁₋₆ alkyl, CF₃, mono-or di-halo-C₁₋₆ alkoxy,        cyano, halo, thioalkyl, hydroxy, alkanoyl, NO₂, SH, amino, C₁₋₆        alkylamino, di (C₁₋₆) alkylamino, di (C₁₋₆) alkylamide,        carboxyl, (C₁₋₆) carboxyester, C₁₋₆ alkylsulfone, C₁₋₆        alkylsulfonamide, di (C₁₋₆) alkyl(alkoxy)amine, C₆₋₁₀ aryl,        C₇₋₁₄ alkylaryl, or a 5-7 membered monocyclic heterocycle;        or a pharmaceutically acceptable enantiomer, diastereomer, salt,        solvate or prodrug thereof.

The present invention also provides compositions comprising thecompounds or pharmaceutically acceptable salts, solvates or prodrugsthereof and a pharmaceutically acceptable carrier. In particular, thepresent invention provides pharmaceutical compositions useful forinhibiting HCV NS3 comprising a therapeutically effective amount of acompound of the present invention, or a pharmaceutically acceptablesalt, solvate or prodrug thereof, and a pharmaceutically acceptablecarrier.

The present invention further provides methods for treating patientsinfected with HCV, comprising administering to the patient atherapeutically effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, solvate or prodrug thereof.Additionally, the present invention provides methods of inhibiting HCVNS3 protease by contacting the NS3 protease with a compound of thepresent invention.

By virute of the present invention, it is now possible to provideimproved drugs comprising the compounds of the invention which can beeffective in the treatment of patients infected with HCV. Specifically,the present invention provides peptide compounds that can inhibit thefunctioning of the NS3 protease, e.g., in combination with the NS4Aprotease. Further, the present invention makes it possible to administercombination therapy to a patient whereby a compound in accordance withthe present invention, which is effective to inhibit the HCV NS3protease, can be administered with another compound having anti-HCVactivity, e.g., a compound which is effective to inhibit the function ofa target selected from the group consisting of HCV metalloprotease, HCVserine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCVentry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH and anucleoside analog for the treatment of an HCV infection.

DETAILED DESCRIPTION OF THE INVENTION

Stereochemical definitions and conventions used herein generally followMcGraw-Hill Dictionary of Chemical Terms, S. P. Parker, Ed., McGraw-HillBook Company, New York (1984) and Stereochemistry of Organic Compounds,Eliel, E. and Wilen, S., John Wiley & Sons, Inc., New York (1994). Manyorganic compounds exist in optically active forms, i.e., they have theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and l or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or l meaning that the compound is levorotatory and (+) or d, meaning thecompound, is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they aremirror images of one another. A specific stereoisomer of a mirror imagepair may also be referred to as an enantiomer, and a mixture of suchisomers is often called an enantiomeric mixture. With reference to theinstances where (R) or (S) is used, it is to designate the absoluteconfiguration of a substituent in context to the whole compound and notin context to the substituent alone.

Unless otherwise specifically noted herein, the terms set forth belowwill have the following definitions.

The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical composition, but differ with regard to the arrangement of theatoms or groups in space.

The term “diastereomer” refers to a stereoisomer which is not anenantiomer, e.g., a stereoisomer with two or more centers of chiralityand whose molecules are not mirror images of one another. Diastereomershave different physical properties, e.g. melting points, boiling points,spectral properties, and reactivities. Mixtures of diastereomers mayseparate under high resolution analytical procedures such aselectrophoresis and chromatography.

The term “enantiomers” refers to two stereoisomers of a compound whichare non-superimposable mirror images of one another.

The term “pharmaceutically acceptable salt” is intended to includenontoxic salts synthesized from a compound which contains a basic oracidic moiety by conventional chemical methods. Generally, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 18th ed., Mack PublishingCompany, Easton, Pa., 1990, p. 1445. The compounds of the presentinvention are useful in the form of the free base or acid or in the formof a pharmaceutically acceptable salt thereof. All forms are within thescope of the invention.

The term “therapeutically effective amount” means the total amount ofeach active component that is sufficient to show a meaningful patientbenefit, e.g., a sustained reduction in viral load. When applied to anindividual active ingredient, administered alone, the term refers tothat ingredient alone. When applied to a combination, the term refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously.

The term “compounds of the invention”, and equivalent expressions, aremeant to embrace compounds of formula I, and pharmaceutically acceptableenantiomer, diastereomer salts, and solvates, e.g. hydrates, andprodrugs. Similarly, references to intermediates, are meant to embracetheir salts, and solvates, where the context so permits. References tothe compound of the invention also include the preferred compounds, e.g.formula II and A-M.

The term “derivative” means a chemically modified compound wherein themodification is considered routine by the ordinary skilled chemist, suchas an ester or an amide of an acid, protecting groups, such as a benzylgroup for an alcohol or thiol, and tert-butoxycarbonyl group for anamine.

The term “solvate” means a physical association of a compound of thisinvention with one or more solvent molecules, whether organic orinorganic. This physical association includes hydrogen bonding. Incertain instances the solvate will be capable of isolation, for examplewhen one or more solvent molecules are incorporated in the crystallattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolable solvates. Exemplary solvates includehydrates, ethanolates, methanolates, isopropanolates and the like.

The term “prodrug” as used herein means derivatives of the compounds ofthe invention which have chemically or metabolically cleavable groupsand become, by solvolysis or under physiological conditions, thecompounds of the invention which are pharmaceutically active in vivo. Aprodrug of a compound may be formed in a conventional manner with afunctional group of the compounds such as with an amino, hydroxy orcarboxy group when present. The prodrug derivative form often offersadvantages of solubility, tissue compatibility, or delayed release in amammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9,21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives wellknown to practitioners of the art, such as, for example, esters preparedby reaction of the parent acidic compound with a suitable alcohol, oramides prepared by reaction of the parent acid compound with a suitableamine.

The term “patient” includes both human and other mammals.

The term “pharmaceutical composition” means a composition comprising acompound of the invention in combination with at least one additionalpharmaceutical carrier, i.e., adjuvant, excipient or vehicle, such asdiluents, preserving agents, fillers, flow regulating agents,disintegrating agents, wetting agents, emulsifying agents, suspendingagents, sweetening agents, flavoring agents, perfuming agents,antibacterial agents, antifungal agents, lubricating agents anddispensing agents, depending on the nature of the mode of administrationand dosage forms. Ingredients listed in Remington's PharmaceuticalSciences, 18^(th) ed., Mack Publishing Company, Easton, Pa. (1999) forexample, may be used.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of patients without excessive toxicity, irritation,allergic response, or other problem or complication commensurate with areasonable risk/benefit ratio.

The term “treating” refers to: (i) preventing a disease, disorder orcondition from occurring in a patient which may be predisposed to thedisease, disorder and/or condition but has not yet been diagnosed ashaving it; (ii) inhibiting the disease, disorder or condition, i.e.,arresting its development; and (iii) relieving the disease, disorder orcondition, i.e., causing regression of the disease, disorder and/orcondition.

The term “substituted” as used herein includes substitution at from oneto the maximum number of possible binding sites on the core, e.g.,organic radical, to which the substituent is bonded, e.g., mono-, di-,tri- or tetra- substituted, unless otherwise specifically stated.

The nomenclature used to describe organic radicals, e.g., hydrocarbonsand substituted hydrocarbons, generally follows standard nomenclatureknown in the art, unless otherwise specifically defined. Combinations ofgroups, e.g., alkylalkoxyamine or arylalkyl, include all possible stableconfigurations, unless otherwise specifically stated. Certain radicalsand combinations are defined below for purposes of illustration.

The term “halo” as used herein means a halogen substituent selected frombromo, chloro, fluoro or iodo. The term “haloalkyl” means an alkyl groupthat in substituted with one or more halo substituents.

The term “alkyl” as used herein means acyclic, straight or branchedchain alkyl substituents having the specified number of carbon atoms andincludes, for example, methyl, ethyl, propyl, butyl, tert-butyl, hexyl,1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl. Thus,C₁₋₆ alkyl refers to an alkyl group having from one to six carbon atoms.The term “lower alkyl” means an alkyl group having from one to six,preferably from one to four carbon atoms. The term “alkylester” means analkyl group additionally containing on ester group. Generally, a statedcarbon number range, e.g., C₂₋₆ alkylester, includes all of the carbonatoms in the radical.

The term “alkenyl” as used herein means an alkyl radical containing atleast one double bond, e.g., ethenyl (vinyl) and alkyl.

The term “alkoxy” as used herein means an alkyl group with the indicatednumber of carbon atoms attached to an oxygen atom. Alkoxy includes, forexample, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and1,1-dimethylethoxy. The latter radical is referred to in the art astert-butoxy. The term “alkoxycarbonyl” means an alkoxy groupadditionally containing a carbonyl group.

The term “cycloalkyl” as used herein means a cycloalkyl substituentcontaining the indicated number of carbon atoms and includes, forexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyland spiro cyclic groups such as spirocyclopropyl as spirocyclobutyl. Theterm “cycloalkoxy” as used herein means a cycloalkyl group linked to anoxygen atom, such as, for example, cyclobutyloxy or cyclopropyloxy. Theterm “alkylcycloalkyl” means a cycloalkyl group linked to an alkylgroup. The stated carbon number range includes the total number ofcarbons in the radical, unless otherwise specfically stated. This aC₄₋₁₀ alkylcycloalkyl may contain from 1-7 carbon atoms in the alkylgroup and from 3-9 carbon atoms in the ring, e.g., cyclopropylmethyl orcyclohexylethyl.

The term “aryl” as used herein means an aromatic moiety containing theindicated number of carbon atoms, such as, but not limited to phenyl,indanyl or naphthyl. For example, C₆₋₁₀ aryl refers to an aromaticmoiety having from six to ten carbon atoms which may be in the form of amonocyclic or bicyclic structure. The term “haloaryl” as used hereinrefers to an aryl mono, di or tri substituted with one or more halogenatoms. The terms “alkylaryl”, “arylalkyl” and “aralalkyl” mean an arylgroup substituted with one or more alkyl groups. Unless the carbon rangeof each group is specified, the stated range applies to the entiresubstituent. Thus, a C₇₋₁₄ alkylaryl group many have from 1-8 carbonatoms in the alkyl group for a monocyclic aromatic and from 1-4 carbonatoms in the alkyl group for a fused aromatic. The attachment of thegroup to bonding site on the molecule can be either at the aryl group orthe alkyl group. Unless a specific aryl radical is specified, e.g.,fluoro-phenyl, or the radical is stated to be unsubstituted, the arylradicals include those substituted with typical substituents known tothose skilled in the art, e.g., halogen, hydroxy, carboxy, carbonyl,nitro, sulfo, amino, cyano, dialkylamino haloalkyl, CF₃, haloalkoxy,thioalkyl, alkanoyl, SH, alkylamino, alkylamide, dialkylamide,carboxyester, alkylsulfone, alkylsulfonamide and alkyl(alkoxy)amine.Examples of alkylaryl groups include benzyl, butylphenyl and1-naphthylmethyl.

The term “alkanoyl” as used herein means straight or branched 1-oxoalkylradicals containing the indicated number of carbon atoms and includes,for example, formyl, acetyl, 1-oxopropyl (propionyl),2-methyl-1-oxopropyl, 1-oxohexyl and the like.

The term “alkylamide” as used herein means an amide mono-substitutedwith an alkyl, such as

The term “heterocycle”, also referred to as “Het”, as used herein means7-12 membered bicyclic heterocycles and 5-9 membered monocyclicheterocycles.

Preferred bicyclic heterocycles are 7-12 membered fused bicyclic ringsystems (both rings share an adjacent pair of atoms) containing from oneto four heteroatoms selected from nitrogen, oxygen and sulfur, whereinone or both rings of the heterocycle can be saturated, partiallysaturated or fully unsaturated ring system (this latter subset alsoherein referred to as unsaturated heteroaromatic). The nitrogen andsulfur heteroatoms atoms may be optionally oxidized. The bicyclicheterocycle may contain the heteroatoms in one or both rings. Unless aspecific heterocycle is specified, e.g., a fluorinated 7-12 memberedbicyclic heterocycle, or the heterocycle is stated to be unsubstituted,the heterocycles include those substituted with typical substituentsknown to those skilled in the art. For example, the bicyclic heterocyclemay also contain substituents on any of the ring carbon atoms, e.g., oneto three substituents. Examples of suitable substituents include C₁₋₆alkyl, C₃₋₇ cycloalkyl, C₁₋₆ alkoxy, C₃₋₇ cycloalkoxy, halo-C₁₋₆ alkyl,CF₃, mono-or di-halo-C₁₋₆ alkoxy, cyano, halo, thioalkyl, hydroxy,alkanoyl, NO₂, SH, amino, C ₁₋₆ alkylamino, di (C₁₋₆) alkylamino, di(C₁₋₆) alkylamide, carboxyl, (C₁₋₆) carboxyester, C₁₋₆ alkylsulfone,C₁₋₆ alkylsulfonamide, C₁₋₆ alkylsulfoxide, di (C₁₋₆)alkyl(alkoxy)amine, C₆₋₁₀ aryl, C₇₋₁₄ alkylaryl, and a 5-7 memberedmonocyclic heterocycle. When two substituents are attached to vicinalcarbon atoms of the bicyclic heterocycle, they can join to form a ring,e.g., a five, six or seven membered ring system containing up to twoheteroatoms selecting from oxygen and nitrogen. The bicyclic heterocyclemay be attached to the molecule, e.g. R₁ in formula I, at any atom inthe ring and preferably carbon.

Examples of bicyclic heterocycles include, but are not limited to, thefollowing ring systems:

Preferred monocyclic heterocycles are 5-9 membered saturated, partiallysaturated or fully unsaturated ring system (this latter subset alsoherein referred to as unsaturated heteroaromatic) containing in the ringfrom one to four heteroatoms selected from nitrogen, oxygen and sulfur,wherein the sulfur and nitrogen heteroatoms may be optionally oxidized.Unless a specific heterocycle is specified, e.g., a C₁₋₆ alkoxysubstituted 5-7 membered monocyclic heterocycle, or the heterocycle isstated to be unsubstituted, the heterocycles include those substitutedwith typical substituents known to those skilled in the art. Forexample, the monocyclic heterocycle may also contain substituents on anyof the ring atoms, e.g., one to three substituents. Examples of suitablesubstituents include C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₁₋₆ alkoxy, C₃₋₇cycloalkoxy, halo-C₁₋₆ alkyl, CF₃, mono-or di-halo-C₁₋₆ alkoxy, cyano,halo, thioalkyl, hydroxy, alkanoyl, NO₂, SH, amino, C₁₋₆ alkylamino, di(C₁₋₆) alkylamino, di (C₁₋₆) alkylamide, carboxyl, (C₁₋₆) carboxyester,C₁₋₆ alkylsulfone, C₁₋₆ alkylsulfoxide, C₁₋₆ alkylsulfonamide, di (C₁₋₆)alkyl(alkoxy)amine, C₆₋₁₀ aryl, C₇₋₁₄ alkylaryl and an additional 5-7membered monocyclic heterocycle. The monocyclic heterocycle may beattached to the molecule, e.g. R₁ in formula I, at any atom in the ring.

Examples of monocyclic heterocycles include, but are not limited to, thefollowing (and their tautomers):

Those skilled in the art will recognize that the heterocycles used inthe compounds of the present invention should be stable. Generally,stable compounds are those which can be synthesized, isolated andformulated using techniques known the those skilled in the art withoutdegradation of the compound.

The term “substituent” with reference to an amino acid or amino acidderivative means a radical derived from the corresponding α-amino acid.For instance, the substituents methyl, iso-propyl, and phenyl representthe amino acids alanine, valine, and phenyl glycine, respectively.

Where used in naming compounds of the present invention, thedesignations “P1′, P1, P2, P3 and P4”, as used herein, map the relativepositions of the amino acid residues of a protease inhibitor bindingrelative to the binding of the natural peptide cleavage substrate.Cleavage occurs in the natural substrate between P1 and P1′ where thenonprime positions designate amino acids starting from the C-terminusend of the peptide natural cleavage site extending towards theN-terminus; whereas, the prime positions emanate from the N-terminus endof the cleavage site designation and extend towards the C-terminus. Forexample, P1′ refers to the first position away from the right hand endof the C-terminus of the cleavage site (i.e. N-terminus first position);whereas P1 starts the numbering from the left hand side of theC-terminus cleavage site, P2: second position from the C-terminus, etc.)[see Berger A. & Schechter I., Transactions of the Royal Society Londonseries (1970), B257, 249-264].

As used herein the term “1-aminocyclopropyl-carboxylic acid” (Acca)refers to a compound of formula:

As used herein the term “tert-butylglycine” refers to a compound of theformula:

The term “residue” with reference to an amino acid or amino acidderivative means a radical derived from the corresponding α-amino acidby eliminating the hydroxyl of the carboxy group and one hydrogen of theα-amino acid group. For instance, the terms Gln, Ala, Gly, Ile, Arg,Asp, Phe, Ser, Leu, Cys, Asn, Sar and Tyr represent the “residues” ofL-glutamine, L-alanine, glycine, L-isoleucine, L-arginine, L-asparticacid, L-phenylalanine, L-serine, L-leucine, L-cysteine, L-asparagine,sarcosine and L-tyrosine, respectively.

The term “side chain” with reference to an amino acid or amino acidresidue means a group attached to the α-carbon atom of the α-amino acid.For example, the R-group side chain for glycine is hydrogen, for alanineit is methyl, for valine it is isopropyl. For the specific R-groups orside chains of the α-amino acids reference is made to A. L. Lehninger'stext on Biochemistry (see chapter 4).

The compounds of the present invention have the structure of Formula I:

wherein:

-   -   (a) R₁ is Het or aryl;    -   (b) m is 1 or 2;    -   (c) n is 1 or 2;    -   (d) R₂ is H; or C₁₋₆ alkyl, C₂₋₆ alkenyl or C₃₋₇ cycloalkyl,        each optionally substituted with halogen;    -   (e) R₃ is C₁₋₈ alkyl optionally substituted with halo, cyano,        amino, C₁₋₆ dialkylamino, C₆₋₁₀ aryl, C₇₋₁₄ alkylaryl, C₁₋₆        alkoxy, carboxy, hydroxy, aryloxy, C₇₋₁₄ alkylaryloxy, C₂₋₆        alkylester or C₈₋₁₅ alkylarylester; C₃₋₁₂ alkenyl, C₃₋₇        cycloalkyl, or C₄₋₁₀ alkylcycloalkyl, wherein the cycloalkyl or        alkylcycloalkyl are optionally substituted with hydroxy, C₁₋₆        alkyl, C₂₋₆ alkenyl or C₁₋₆ alkoxy; or R₃ together with the        carbon atom to which it is attached forms a C₃₋₇ cycloalkyl        group optionally substituted with C₂₋₆ alkenyl;    -   (f) Y is H, phenyl substituted with nitro, pyridyl substituted        with nitro, or C₁₋₆ alkyl optionally substituted with cyano, OH        or C₃₋₇ cycloalkyl; provided that if R₄ or R₅ is H then Y is H;    -   (g) B is H, C₁₋₆ alkyl, R₄—(C═O)—, R₄O(C═O)—, R₄—N(R₅)—C(═O)—,        R₄—N(R₅)—C(═S)—, R₄SO₂—, or R₄—N(R₅)—SO₂—;    -   (h) R₄ is (i) C₁₋₁₀ alkyl optionally substituted with phenyl,        carboxyl, C₁₋₆ alkanoyl, 1-3 halogen, hydroxy, —OC(O)C₁₋₆ alkyl,        C₁₋₆ alkoxy, amino optionally substituted with C₁₋₆ alkyl,        amido, or (lower alkyl) amido; (ii) C₃₋₇ cycloalkyl, C₃₋₇        cycloalkoxy, or C₄₋₁₀ alkylcycloalklyl, each optionally        substituted with hydroxy, carboxyl, (C₁₋₆ alkoxy)carbonyl, amino        optionally substituted with C₁₋₆ alkyl, amido, or (lower alkyl)        amido; (iii) C₆₋₁₀ aryl or C₇₋₁₆ arylalkyl, each optionally        substituted with C₁₋₆ alkyl, halogen, nitro, hydroxy, amido,        (lower alkyl) amido, or amino optionally substituted with C₁₋₆        alkyl; (iv) Het; (v) bicyclo(1.1.1)pentane; or (vi) —C(O)OC₁₋₆        alkyl, C₂₋₆alkenyl or C₂₋₆ alkynyl;    -   (i) R₅ is H; C₁₋₆ alkyl optionally substituted with 1-3        halogens; or C₁₋₆ alkoxy provided R₄ is C₁₋₁₀ alkyl;    -   (j) X is O, S, SO, SO₂, OCH₂, CH₂O or NH;    -   (k) R′ is Het, C₆₋₁₀ aryl or C₇₋₁₄ alkylaryl, each optionally        substituted with R^(a); and    -   (l) R^(a) is C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₁₋₆ alkoxy, C₃₋₇        cycloalkoxy, halo-C₁₋₆ alkyl, CF₃, mono-or di-halo-C₁₋₆ alkoxy,        cyano, halo, thioalkyl, hydroxy, alkanoyl, NO₂, SH, amino, C₁₋₆        alkylamino, di (C₁₋₆) alkylamino, di (C₁₋₆) alkylamide,        carboxyl, (C₁₋₆) carboxyester, C₁₋₆ alkylsulfone, C₁₋₆        alkylsulfonamide, di (C₁₋₆) alkyl(alkoxy)amine, C₆₋₁₀ aryl,        C₇₋₁₄ alkylaryl, or a 5-7 membered monocyclic heterocycle;        or a pharmaceutically acceptable enantiomer, diastereomer, salt,        solvate or prodrug thereof.

Preferably, Het is unsubstituted Het or Het substituted with from one tothree of halo, cyano, trifluoromethyl, nitro, C₁₋₆ alkyl, C₁₋₆ alkoxy,amido, C₁₋₆ alkanoylamino, amino, phenyl or phenylthio, said phenyl orphenyl portion of phenylthio being unsubstituted or substituted with oneto three substituents selected from halo, cyano, nitro, C₁₋₆ alkyl, C₁₋₆alkoxy, amido or phenyl;

More preferably, R₁ is selected from the group consisting of

In another aspect of the invention, R₁ is a monocyclic aryl, preferablya C₆ aryl, optionally substituted with halo, e.g., chloro, hydroxyl,C₁₋₆ alkoxy, e.g., methoxy, or haloC₁₋₆ alkoxy, e.g., trifluoromethoxy.

Preferably, R₂ is C₁₋₆ alkyl, C₂₋₆ alkenyl or C₃₋₇ cycloalkyl. Morepreferably, R₂ is C₂₋₆ alkenyl. Preferably, R₃ is C₁₋₈ alkyl optionallysubstituted with C₆aryl, C₁₋₆ alkoxy, carboxy, hydroxy, aryloxy, C₇₋₁₄alkylaryloxy, C₂₋₆ alkylester or C₈₋₁₅ alkylarylester; C₃₋₁₂ alkenyl;C₃₋₇ cycloalkyl; or C₄₋₁₀ alkylcycloalkyl. More preferably, R₃ is C₁₋₈alkyl optionally substituted with C₁₋₆ alkoxy; or C₃₋₇ cycloalkyl.

Preferably, Y is H. Preferably, B is H, C₁₋₆ alkyl, R₄—(C═O)—,R₄O(C═O)—, R₄—N(R₅)—C(═O)—, R₄—N(R₅)—C(═S)—, R₄SO₂—, or R₄—N(R₅)—SO₂—.More preferably, B is R₄—(C═O)—, R₄O(C═O)—, or R₄-N(R₅)—C(═O)—. Evenmore preferably, B is R₄O(C═O)— and R₄is C₁₋₆alkyl. Preferably, R₄ is(i) C₁₋₁₀ alkyl optionally substituted with phenyl, carboxyl, C₁₋₆alkanoyl, 1-3 halogen, hydroxy, C₁₋₆ alkoxy; (ii) C₃₋₇ cycloalkyl, C₃₋₇cycloalkoxy, or C₄₋₁₀ alkylcycloalklyl; or (iii) C₆₋₁₀ aryl or C₇₋₁₆arylalkyl, each optionally substituted with C₁₋₆ alkyl or halogen. Morepreferably, R₄ is (i) C₁₋₁₀ alkyl optionally substituted with 1-3halogen or C₁₋₆ alkoxy; or (ii) C₃₋₇ cycloalkyl or C₄₋₁₀alkylcycloalkyl. Preferably, R₅is H or C₁₋₆ alkyl optionally substitutedwith 1-3 halogens. More preferably, R₅ is H.

Preferably, X is O or NH. Preferably, R′ is Het or C₆₋₁₀ aryl eachoptionally substituted with R^(a). More preferably, R′ is Het.Preferably, the heterocycle contains 1 or 2 nitrogen atoms andoptionally a sulfur atom or an oxygen atom in the ring. More preferably,the heterocycle is substituted with at least one of C₁₋₆ alkyl, C₁₋₆alkoxy, halo, C₆₋₁₀ aryl, C₇₋₁₄ alkylaryl, or a 5-7 membered monocyclicheterocycle. Preferably, R^(a) is C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₁₋₆alkoxy, halo-C₁₋₆ alkyl, halo, amino, C₆ aryl, or a 5-7 memberedmonocyclic heterocycle.

The substituents from each grouping may be selected individually andcombined in any combination which provides a stable compound inaccordance with the present invention. Also, more than one substituentfrom each group may be substituted on the core group provided there aresufficient available binding sites. For example, each of the followingR₆, R₇, R₈ or R₉ substituents, C₁₋₆ alkoxy, C₆ aryl and a 5-7 memberedmonocyclic heterocycle, may be substituted on a bicyclic heterocycle.

In a preferred aspect, the compounds of the present invention have thestructure of Formula II:

wherein:

-   -   (a) R₁ is unsubstituted Het or Het substituted with from one to        three of halo, cyano, trifluoromethyl, C₁₋₆ alkyl, C₁₋₆ alkoxy,        amido, C₁₋₆ amino, phenyl, said phenyl being unsubstituted or        substituted with one to three substituents selected from halo,        cyano, nitro, C₁₋₆ alkyl, C₁₋₆ alkoxy, amido or phenyl; or C₆        aryl;    -   (b) R₂ is C₁₋₆ alkyl, C₂₋₆ alkenyl or C₃₋₇ cycloalkyl;    -   (c) R₃ is C₁₋₈ alkyl optionally substituted with C₆ aryl, C₁₋₆        alkoxy, carboxy, hydroxy, aryloxy, C₇₋₁₄ alkylaryloxy, C₂₋₆        alkylester, C₈₋₁₅ alkylarylester; C₃₋₁₂ alkenyl, C₃₋₇        cycloalkyl, or C₄₋₁₀ alkylcycloalkyl;    -   (d) B is H, C₁₋₆ alkyl, R₄—(C═O)—, R₄O (C═O)—, R₄—N(R₅)—C(═O)—,        R₄—N(R₅)—C(═S)—, R₄SO₂—, or R₄—N(R₅)—SO₂—;    -   (e) R₄ is (i) C₁₋₁₀ alkyl optionally substituted with phenyl,        carboxyl, C₁₋₆ alkanoyl, 1-3 halogen, hydroxy, C₁₋₆ alkoxy; (ii)        C₃₋₇ cycloalkyl, C₃₋₇ cycloalkoxy, or C₄₋₁₀ alkylcycloalklyl;        or (iii) C₆₋₁₀ aryl or C₇₋₁₆ arylalkyl, each optionally        substituted with C₁₋₆ alkyl or halogen;    -   (f) R₅ is H or C₁₋₆ alkyl optionally substituted with 1-3        halogens;    -   (g) X is O or NH;    -   (h) R′ is Het; or C₆₋₁₀ aryl optionally substituted with R^(a);        and    -   (i) R^(a) is C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₁₋₆ alkoxy, halo-C₁₋₆        alkyl, halo, amino, C₆ aryl, or a 5-7 membered monocyclic        heterocycle;        or a pharmaceutically acceptable enantiomer, diastereomer, salt,        solvate or prodrug thereof.

In one aspect of the invention, R′ is a bicyclic heterocycle.Preferably, the heterocycle contains 1 or 2 nitrogen atoms andoptionally a sulfur atom or an oxygen atom in the ring. More preferably,the heterocycle is substituted with at least one of C₁₋₆ alkyl, C₁₋₆alkoxy, halo, C₆ aryl, and a 5-7 membered monocyclic heterocycle. Evenmore preferably, R′ is a bicyclic heterocycle containing I nitrogen atomand substituted with methoxy and at least one of a C₆ aryl and a 5-7membered monocyclic heterocycle.

In another aspect of the invention, R′ is a monocyclic heterocycle.Preferably, the heterocycle contains 1 or 2 nitrogen. atoms andoptionally a sulfur atom or an oxygen atom in the ring. More preferably,the heterocycle is substituted with at least one of C₁₋₆ alkyl, C₁₋₆alkoxy, halo, C₆₋₁₀ aryl, C₇₋₁₄ alkylaryl, or a 5-7 membered monocyclicheterocycle. Even more preferably, R′ is a monoyclic heterocyclecontaining 1 or 2 nitrogen atoms and substituted with methoxy and atleast one of a C₆ aryl and a 5-7 membered monocyclic heterocycle.

In a more preferred aspect of the invention, the compounds have thestructure of Formula III

wherein:

-   -   (a) R₁ is unsubstituted Het or Het substituted with one to three        of halo, cyano, trifluoromethyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, amido,        or amino; or a C₆ aryl;    -   (b) R₂ is C₂₋₆ alkenyl;    -   (c) R₃ is C₈ alkyl;    -   (d) B is R₄O(C═O)—, or R₄—N(H)—C(═O)—;    -   (e) R₄ is C₁₋₁₀ alkyl;    -   (f) R′ is a bicyclic heterocycle optionally substituted with        R^(a); and    -   (g) R^(a) is C₁₋₆ alkyl, C₁₋₆ alkoxy, halo, C₆ aryl, or a 5-7        membered monocyclic heterocycle;        or a pharmaceutically acceptable enantiomer, diastereomer, salt,        solvate or prodrug thereof.

Preferably, R₂ is vinyl, R₃ is t-butyl, R₄ is t-butyl, R′ is quinolineor isoquinoline optionally substituted with R^(a). Preferably, R^(a) isC₁₋₆ alkoxy. More preferably, R^(a) further includes at least one of C₆aryl or a 5-7 membered monocyclic heterocycle.

The compounds of the present invention can be manufactured by methodsknown to those skilled in the art, see e.g., U.S. Pat. No. 6,323,180 andU.S. patent application Ser. No. 20020111313 A1. The following methodsset forth below are provided for illustrative purposes and are notintended to limit the scope of the claimed invention. It will berecognized that it may be preferred or necessary to prepare such acompound in which a functional group is protected using a conventionalprotecting group then to remove the protecting group to provide acompound of the present invention. The details concerning the use ofprotecting groups in accordance with the present invention are known tothose skilled in the art.

The compounds of the present invention may, for example, be synthesizedaccording to a general process as illustrated in Scheme I (wherein CPGis a carboxyl protecting group and APG is an amino protecting group):

Briefly, the P1, P2, and P3 can be linked by well known peptide couplingtechniques. The P1, P2, and P3 groups may be linked together in anyorder as long as the final compound corresponds to peptides of theinvention. For example, P3 can be linked to P2-P1; or P1 linked toP3-P2.

Generally, peptides are elongated by deprotecting the α-amino group ofthe N-terminal residue and coupling the unprotected carboxyl group ofthe next suitably N-protected amino acid through a peptide linkage usingthe methods described. This deprotection and coupling procedure isrepeated until the desired sequence is obtained. This coupling can beperformed with the constituent amino acids in stepwise fashion, asdepicted in Scheme I.

Coupling between two amino acids, an amino acid and a peptide, or twopeptide fragments can be carried out using standard coupling proceduressuch as the azide method, mixed carbonic-carboxylic acid anhydride(isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide,diisopropylcarbodiimide, or water-soluble carbodiimide) method, activeester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method,Woodward reagent K-method, carbonyldiimidazole method, phosphorusreagents or oxidation-reduction methods. Some of these methods(especially the carbodiimide method) can be enhanced by adding1-hydroxybenzotriazole or 4-DMAP. These coupling reactions can beperformed in either solution (liquid phase) or solid phase.

More explicitly, the coupling step involves the dehydrative coupling ofa free carboxyl of one reactant with the free amino group of the otherreactant in the present of a coupling agent to form a linking amidebond. Descriptions of such coupling agents are found in generaltextbooks on peptide chemistry, for example, M. Bodanszky, “PeptideChemistry”, 2^(nd) rev ed., Springer-Verlag, Berlin, Germany, (1993).Examples of suitable coupling agents are N,N′-dicyclohexylcarbodiimide,1-hydroxybenzotriazole in the presence of N,N′-dicyclohexylcarbodiimideor N-ethyl-N′-[(3-dimethylamino)propyl]carbodiimide. A practical anduseful coupling agent is the commercially available(benzotriazol-1-yloxy)tris-(dimethylamino)phosphoniumhexafluorophosphate, either by itself or in the present of1-hydroxybenzotriazole or 4-DMAP. Another practical and useful couplingagent is commercially available2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate.Still another practical and useful coupling agent is commerciallyavailable O-(7-azabenzotrizol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate. The coupling reaction is conducted in an inertsolvent, e.g. dichloromethane, acetonitrile or dimethylformamide. Anexcess of a tertiary amine, e.g. diisopropylethylamine,N-methylmorpholine, N-methylpyrrolidine or 4-DMAP is added to maintainthe reaction mixture at a pH of about 8. The reaction temperatureusually ranges between 0° C. and 50° C. and the reaction time usuallyranges between 15 min and 24 h.

The functional groups of the constituent amino acids generally must beprotected during the coupling reactions to avoid formation of undesiredbonds. Protecting groups that can be used are listed, for example, inGreene, “Protective Groups in Organic Chemistry”, John Wiley & Sons, NewYork (1981) and “The Peptides: Analysis, Synthesis, Biology”, Vol. 3,Academic Press, New York (1981), the disclosures of which are herebyincorporated by reference.

The α-amino group of each amino acid to be coupled to the growingpeptide chain must be protected (APG). Any protecting group known in theart can be used. Examples of such groups include: 1) acyl groups such asformyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl; 2) aromaticcarbamate groups such as benzyloxycarbonyl (Cbz or Z) and substitutedbensyloxycarbonyls, and 9-fluorenylmethyloxycarbonyl (Fmoc); 3)aliphatic carbamate groups such as tert-butyloxycarbonyl (Boc),ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4)cyclic alkyl carbamate groups such as cyclopentyloxycarbonyl andadamantyloxycarbonyl; 5) alkyl groups such as triphenylmethyl andbenzyl; 6)trialkylsilyl such as trimethylsilyl; and 7) thiol containinggroups such as phenylthiocarbonyl and dithiasuccinoyl.

The preferred α-amino protecting group is either Boc or Fmoc. Many aminoacid derivatives suitably protected for peptide synthesis arecommercially available. The α-amino protecting group of the newly addedamino acid residue is cleaved prior to the coupling of the next aminoacid. When the Boc group is used, the methods of choice aretrifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane orin ethyl acetate. The resulting ammonium salt is then neutralized eitherprior to the coupling or in situ with basic solutions such as aqueousbuffers, or tertiary amines in dichloromethane or acetonitrile ordimethylformamide. When the Fmoc group is used, the reagents of choiceare piperidine or substituted piperidine in dimethylformamide, but anysecondary amine can be used. The deprotection is carried out at atemperature between 0° C. and room temperature (rt or RT) usually 20-22°C.

Any of the amino acids having side chain functionalities must beprotected during the preparation of the peptide using any of theabove-described groups. Those skilled in the art will appreciate thatthe selection and use of appropriate protecting groups for these sidechain functionalities depend upon the amino acid and presence of otherprotecting groups in the peptide. The selection of such protectinggroups is important in that the group must not be removed during thedeprotection and coupling of the α-amino group.

For example, when Boc is used as the α-amino protecting group, thefollowing side chain protecting group are suitable: p-toluenesulfonyl(tosyl) moieties can be used to protect the amino side chain of aminoacids such as Lys and Arg; acetamidomethyl, benzyl (Bn), ortert-butylsulfonyl moieties can be used to protect the sulfidecontaining side chain of cysteine; bencyl (Bn) ethers can be used toprotect the hydroxy containing side chains of serine, threonine orhydroxyproline; and benzyl esters can be used to protect the carboxycontaining side chains of aspartic acid and glutamic acid.

When Fmoc is chosen for the α-amine protection, usually tert-butyl basedprotecting groups are acceptable. For instance, Boc can be used forlysine and arginine, tert-butyl ether for serine, threonine andhydroxyproline, and tert-butyl ester for aspartic acid and glutamicacid. Triphenylmethyl (Trityl) moiety can be used to protect the sulfidecontaining side chain of cysteine.

Once the elongation of the peptide is completed all of the protectinggroups are removed. When a liquid phase synthesis is used, theprotecting groups are removed in whatever manner is dictated by thechoice of protecting groups. These procedures are well known to thoseskilled in the art.

Further, the following guidance may be followed in the preparation ofcompounds of the present invention. For example, to form a compoundwhere R₄—C(O)—, R₄—S(O)₂, a protected P3 or the whole peptide or apeptide segment is coupled to an appropriate acyl chloride or sulfonylchloride respectively, that is either commercially available or forwhich the synthesis is well known in the art. In preparing a compoundwhere R₄O—C(O)—, a protected P3 or the whole peptide or a peptidesegment is coupled to an appropriate chloroformate that is eithercommercially available or for which the synthesis is well known in theart. For Boc-derivatives (Boc)₂O is used.

For example:

Cyclopentanol is treated with phosgene to furnish the correspondingchloroformate.

The chloroformate is treated with the desired NH₂-tripeptide in thepresence of a base such as triethylamine to afford thecyclopentylcarbamate.

In preparing a compound where R₄—N(R₅)—C(O)—, or R₄—NH—C(S)—, aprotected P3 or the whole peptide or a peptide segment is treated withphosgene followed by amine as described in SynLett. February 1995; (2);142-144 or is reacted with the commercially available isocyanate and asuitable base such as triethylamine.

In preparing a compound where R₄—N(R₅)—S(O₂), a protected P3 or thewhole peptide or a peptide segment is treated with either a freshlyprepared or commercially available sulfamyl chloride followed by amineas described in patent Ger. Offen.(1998), 84 pp. DE 19802350 or WO98/32748.

The α-carboxyl group of the C-terminal residue is usually protected asan ester (CPG) that can be cleaved to give the carboxylic acid.Protecting groups that can be used include: 1) alkyl esters such asmethyl, trimethylsilylethyl and t-butyl, 2) aralkyl esters such asbenzyl and substituted benzyl, or 3) esters that can be cleaved by mildbase treatment or mild reductive means such as trichloroethyl andphenacyl esters.

The resulting a-carboxylic acid (resulting from cleavage by mild acid,mild base treatment or mild reductive means) is coupled with a R₁SO₂NH₂.

Compounds of the present invention can be prepared by many methodsincluding those described in the examples, below, and as described inU.S. Pat. No. 6,323,180 and U.S. patent application Ser. No. 10/001,850filed on Nov. 20, 2001. The teachings of U.S. Pat. No. 6,323,180 andU.S. patent application Ser. No. 10/001,850 are incorporated herein, intheir entirety, by reference.

Scheme II further shows the general process wherein compounds of FormulaI are constructed by the coupling of tripeptide carboxylic acidintermediate (1) with a P1′ sulfonamide. (It should be noted that thegroups R₆, R₇, R₈, R₉, R₁₀, R₁₁ as shown below represent substituents ofthe heterocyclic system.) Said coupling reaction requires treatment ofcarboxylic acid (1) with a coupling reagent such as carbonyl diimidazolein a solvent such as THF, which can be heated to reflux, followed by theaddition of the formed derivative of (1), to the P1′ sulfonamide, in asolvent such as THF or methylene chloride in the presence of a base suchas DBU.

An alternative process for the construction of compounds of Formula I isshown in Scheme III. Therein the P1′ sulfonamide element is coupled tothe P1 element using the process employed in Scheme 1. The resultingP1-P1′ moiety can then be deprotected at it's amino terminus. In thisgeneral example a Boc protecting group is employed but one skilled inthe art would recognize that a number of suitable amino protectinggroups could be employed in this process. Said Boc protecting group canbe removed using acid such as trifluoroacetic acid in a solvent such asdichloroethane to provide the deprotected amine as the TFA salt. SaidTFA amine salt can be directly employed in the subsequent couplingreaction or as an alternative the TFA amine salt can be first convertedto the HCl amine salt, and this HCl amine salt is used in said couplingreaction as shown in Scheme III. The coupling of said HCl amine salt (3)with the carboxyl terminus a P4-P3-P2 intermediate can be achieved usingcoupling reagents, such as HATU, in solvents such as dichloromethane toprovide compounds of Formula I (4).

An alternative process for the construction of compounds of Formula I isshown in Scheme IV. Herein the hydrochloride salt of the P1-P1′ terminalamine (1) is coupled to the free carboxyl group of the P2 element usingcoupling agents such as PyBOP, in the presence of a base such asdiisopropyl amine, and in a solvent such as methylene chloride. Theresulting P2-P1-P1′ intermediate can be converted to compounds ofFormula I in a two step process wherein the first step is deprotectionof the P2 amine terminus using an acid such as TFA in a solvent such asmethylene chloride. The resulting trifluoroacetic acid salt can becoupled with the carboxyl terminus of the P4-P3 element using standardcoupling agents such as PyBop in the presence of base such asdiisopropyl amine, and using solvents such methylene chloride to providecompounds of Formula I (4).

The P4-P3-P2 intermediate utilized in the above schemes can beconstructed as previously described with a further description of thisprocess shown in general Scheme V. Therein the free carboxyl terminus ofthe P4-P3 intermediate (1), can be coupled to the amino terminus of theP2 element to provide the P4-P3-P2 dipeptide (2). The carboxyl terminusof the P4-P3-P2 intermediate can be deprotected by saponification of theester group to provide P4-P3-P2 as the free carboxylic acid (3).Intermediates like (3) can be converted to compounds of Formula I usingthe methods described herein.

Compounds of Formula 1 can also be converted into other compounds ofFormula I as described herein. An example of such a process is shown inScheme VI wherein a compound of Formula I (1) which bears a Boc group atthe P4 position is converted in a compound of Formula I (3) wherein saidcompound bears a urea group at the P4 position. The conversion of (1) to(3) can be carried out in a two step process the first of which is theconversion of (1) to amine (2) by treatment of (1) with an acid such asTFA in a solvent such as methylene chloride. The resulting amine TFAsalt can be treated with an isocyanate in the presence of one equivalentof base to provide a compound of Formula I (3) wherein the P3 moiety iscapped with a urea. As previously noted one skilled in the art willrecognize that intermediate (2) can be used as starting materials forthe preparation of compounds of Formula I wherein the P3 group is cappedwith an amide or a sulfonamide, or thiourea, or a sulfamide. Theconstruction of said compounds of Formula I can be achieved usingstandard conditions for the formation of said P4 functionalities fromamines.

In the construction of compounds of Formula I, the P1′ terminus isincorporated into the molecules using one of the processes outlinedabove and described in more detail below. In some examples the P1′elements, that is the heterocyclesulfonamides are commercially availableor can be prepared as described herein.

The P1 elements utilized in generating compounds of Formula I are insome cases commercially available, but are otherwise synthesized usingthe methods described herein and subsequently incorporated intocompounds of Formula I using the methods described herein. Thesubstituted P1 cyclopropylamino acids can be synthesized following thegeneral process outline in Scheme VIII.

Treatment of commercially available or easily synthesized imine (1) with1,4-dihalobutene (2) in presence of a base produces, provides theresulting imine (3). Acid hydrolysis of 3 then provides 4, which has anallyl substituent syn to the carboxyl group as a major product. Theamine moiety of 4 can protected using a Boc group to provide the fullyprotected amino acid 5. This intermediate is a racemate which can beresolved by an enzymatic process wherein the ester moiety of 5 iscleaved by a protease to provide the corresponding carboxylic acid.Without being bound to any particular theory, it is believed that thisreaction is selective in that one of the enantiomers undergoes thereaction at a much greater rate than its mirror image providing for akinetic resolution of the intermediate racemate. In the examples citedherein, the more preferred stereoisomer for integration into compoundsof Formula I is 5a which houses the (1R, 2S) stereochemistry. In thepresence of the enzyme, this enantiomer does not undergo ester cleavageand thereby this enantiomer 5a is recovered from the reaction mixture.However, the less preferred enantiomer ,5b with houses the (1S, 2R)stereochemistry undergoes ester cleavage, i.e., hydrolysis, to providethe free acid 6. Upon completion of this reaction, the ester 5a can beseparated from the acid product 6 by routine methods such as, forexample, aqueous extraction methods or chromatography.

Procedures for making P2 intermediates and compounds of Formula I areshown in the Schemes below. It should be noted that in many casesreactions are depicted for only one position of an intermediate.However, it is to be understood that such reactions could be used toimpart modifications to other positions within this intermediate.Moreover, said intermediates, reaction conditions and methods given inthe specific examples are broadly applicable to compounds with othersubstitution patterns. The general Schemes outlined below are followedwith examples herein. Both general and specific examples arenon-limiting, as for example the isoquinoline nucleus is shown as partof the general scheme, Scheme IX, however, this pathway represents aviable process for the construction of alternate heterocyclesubstituents as replacements for the isoquinoline element, such asquinolines, or pyridines.

Scheme IX shows the coupling of an N-protected C4-hydroxyproline moietywith a heterocycle to form intermediate (4) and the subsequentmodification of said intermediate (4) to a compound of Formula I by theprocess of peptide elongation as described herein. It should be notedthat in the first step, that is the coupling of the C4-hydroxy prolinegroup with the heteroaryl element, a base is employed. One skilled inthe art would recognized that this coupling can be done using bases suchas potassium tert-butoxide, or sodium hydride, in solvent such as DMF orDMSO or THF. This coupling to the isoquinoline ring system occurs at theC1 position (numbering for isoquinoline ring system shown inintermediate 2 of Scheme IX) and is directed by the chloro group whichis displaced in this process. It should be noted that the alternativeleaving groups can be utilized at this position such as a fluoro asshown in the Scheme. Said fluoro intermediates (3) are available fromthe corresponding chloro compound using literature procedures describedherein. It should also be noted that the position of the leaving group(chloro or fluoro) in a given ring system can vary as shown in Scheme X,wherein the leaving group (fluoro in this example) is in the C6 positionof the isoquinoline ring system of intermediate (2).

It should be further noted that the position of the ring heteroatom(s)in intermediates like (2) of Scheme IX and Scheme X is also variable, asdefined by the term heterocycle described herein. In Scheme Xintermediate (2) can be coupled to a C4 hydroxy proline derivative toprovide the P2 element (3). This C6-substituted isoquinoline derivativecan be converted to compounds of Formula I using the methods describedherein.

An alternative to the method described above for the coupling of theC4-hydroxyproline to aromatics and heteroaromatics, is provided in theMitsunobu reaction as depicted in

step 1 of Scheme XI. In this general reaction Scheme a C4-hydroxyproline derivative is coupled to a quinazoline ring system. Thisreaction makes use of reagents such as triphenylphosphine and DEAD(diethylazodicarboxylate) in aprotic solvents such as THF or dioxane andcan be used for the formation of aryl and heteroaryl ethers. Note thatin the course of this coupling reaction the stereochemistry of the C4chiral center in the C4-hydroxyproline derivative is inverted andthereby it is necessary to use the C4-hydroxyproline derivative housingthe (S) stereochemistry at the C4 position as starting material. (asshown in Scheme XI). It should be noted that numerous modifications andimprovements of the Mitsunobu reaction have been described in theliterature, the teachings of which are incorporated herein.

In a subset of examples herein, isoquinolines are incorporated into thefinal compounds and specifically into the P2 region of said compounds.One skilled in the art would recognize that a number of general methodsare available for the synthesis of isoquinolines. Moreover, saidisoquinolines generated by these methods can be readily incorporatedinto final compounds of Formula I using the processes described herein.One general methodology for the synthesis of isoquinolines is shown inScheme XII, wherein cinnamic acid derivatives, shown in general form asstructure (2) are

converted to 1-chloroisoquinolines in a four step process. Saidchloroisoquinolines can be subsequently used in coupling reactions toC4-hydroxyproline derivatives as described herein. The conversion ofcinnamic acids to chloroquinolines begins with the treatment of cinnamicacid with an alkylcholorformate in the presence of a base. The resultinganhydride is then treated with sodium azide which results in theformation of an acylazide (3) as shown in the Scheme. Alternate methodsare available for the formation of acylazides from carboxylic acids asfor example said carboxylic acid can be treated withdiphenylphosphorylazide (DPPA) in an aprotic solvent such as methylenechloride in the presence of a base. In the next step of the reactionsequence the acylazide (3) is converted to the correspondingisoquinolone (4) as shown in the Scheme. In the event the acylazide isheated to a temperature of approximately 190 degrees celcius in a highboiling solvent such a diphenylmethane. This reaction is general andprovides moderate to good yields of substituted isoquinolone from thecorresponding cinnamic acid derivatives. It should noted that saidcinnamic acid derivatives are available commercially or can be obtainedfrom the corresponding benzaldehyde (1) derivative by directcondensation with malonic acid or derivatives thereof and also byemploying a Wittig reaction. The intermediate isoquinolones (4) ofScheme XII can be converted to the corresponding 1-chloroisoquinoline bytreatment with phosphorous oxychloride. This reaction is general and canbe applied to any of the isoquinolones, quinolones or additionalheterocycles as shown herein to covert a hydroxy substituent to thecorresponding chloro compound when said hydroxy is in conjugation with anitrogen atom in said heterocylic ring systems.

An alternative method for the synthesis of the isoquinoline ring systemis the Pomeranz-Fritsh procedure. This general method is outlined inScheme XIII. The process begins with the conversion of a benzaldehydederivative (1) to a functionalized imine (2). Said imine is thenconverted to the isoquinoline ring system by treatment with acid atelevated

temperature. This isoquinoline synthesis of Scheme XIII is general, andit should be noted that this process is particularly useful in procuringisoquinoline intermediates that are substituted at the C8 position(note: in intermediate (3) of Scheme XIII the C8 position of theisoquinoline ring is substituted with substituent R₁₁). The intermediateisoquinolines (3) can be converted to the corresponding1-chloroquinolines (5) in a two step process as shown. The first step inthis sequence is the formation of the isoquinoline N-oxide(4) bytreatment of isoquinoline (3) with meta-chloroperbenzoic acid in anaprotic solvent such as dichloromethane. Intermediate (4) can beconverted to the corresponding 1-chloroquinoline by treatment withphosphorous oxychloroide in refluxing chloroform. Note this two stepprocess is general and can be employed to procure chloroisoquinolinesand chloroquinolines from the corresponding isoquinolines and quinolinesrespectively. Another method for the synthesis of the isoquinoline ringsystem is shown in Scheme XIV. In this process an ortho-alkylbenzamidederivative (1) is treated with a strong

base such as tert-butyl lithium in a solvent such as THF at lowtemperature. To this reaction mixture is then added a nitrilederivative, which undergoes an addition reaction with the anion derivedfrom deprotonation of (1), resulting in the formation of (2). Thisreaction is general and can be used for the formation of substitutedisoquinolines. Intermediate (2) of Scheme XIV can be converted to thecorresponding 1-chloroquinoline by the methods described herein.

An additional method for the synthesis of isoquinolines is shown inScheme XV. The deprotonation of intermediate (1) using tert-butyllithium is described above. In the present method however, saidintermediate anion is trapped by an ester, resulting in the formation ofintermediate (2) as shown below. In a subsequent reaction ketone (2) iscondensed with ammonium acetate at elevated temperature providing forthe formation of quinolone (3). This reaction is general and can beapplied for the construction of substituted isoquinolones which can thenbe converted to the corresponding 1-chloroisoquinolines as describedherein.

Yet an additional method for the construction of isoquinolines is foundin Scheme XVI. In the first step of this process an ortho-alkylaryliminederivatives such as (1) is subjected to deprotonation conditions(sec-butyl lithium, THF) and the resulting anion is quenched by

the addition of an activated carboxylic acid derivative such as aWeinreb amide. The resulting keto imine (2) can be converted to thecorresponding isoquinoline by condensation with ammonium acetate atelevated temperatures. This method is general and can be used for thesynthesis of substituted isoquinolines. Said isoquinolines can beconverted to the corresponding 1-chloroquinoline by the methodsdescribed herein.

The heterocycles described herein, and which are incorporated into thecompounds of Formula I can be further functionalized. It is obvious toone skilled in the art that additional functionalization of saidheterocycles can be done either before or after incorporation of thesefunctionalities into compounds of Formula I. The following Schemesillustrate this point. For example Scheme XVII shows the conversion of a1-chloro-

6-fluoro-isoquinoline to the corresponding1-chloro-6-alkoxy-isoquinoline species, by treatment of (1) of (eq. 1)with a sodium or potassium alkoxide species in the alcohol solvent fromwhich the alkoxide is derived at room temperature. In some cases it maybe necessary to heat the reaction to drive it to completion. Saidchloroquinoline can be incorporated into a compound of Formula I usingthe art described herein. Modifications of a P2 heterocyclic element canalso be done after it's incorporation into compounds of Formula I asshown in (eq. 2) of Scheme VXII. Specifically compounds such as (1) in(eq. 2) which contain a leaving group in the pthalazine nucleus can bedisplaced by a nucleophile such as an alkoxide in solvents such as thecorresponding alcohol from which the alkoxide is derived. These reactionscan be conducted at room temperature but in some cases it may benecessary to heat the reaction to drive it to completion.

Scheme XVIII provides a general example for the modification ofheterocycles as defined herein by employing palladium mediated couplingreactions. Said couplings can be employed to functionalize a heterocycleat each position of the ring system providing said ring is suitablyactivated or functionalized, as for example with a chloride as shown inthe Scheme. This sequence begins with 1-chloroisoquinoline (1) whichupon treatment with metachloroperbenzoic acid can be converted to thecorresponding N-oxide (2). Said intermediate (2) can be converted to thecorresponding 1,3-dichloroisoquinoline (3) by treatment with phosphorousoxychloride in refluxing chloroform. Intermediate (3) can be coupledwith N-Boc-4-hydroxyproline by the methods described herein to provideintermediate (5) as shown in the Scheme. Intermediate (5) can undergo aSuzuki coupling with an aryl boronic acid, in the presence of apalladium reagent and base, and in a solvent such as THF or toluene orDMF to provide the C3-arylisoquinoline intermediate (6).Heteroarylboronic acids can also be employed in this Pd mediatedcoupling process to provide C3-heteroarylisoquinolines. Intermediate (6)can be converted into final compounds of Formula I by the methodsdescribed herein.

Palladium mediated couplings of heteroaryl systems with aryl orheteroaryl elements can also be employed at a later synthetic stage inthe construction of compounds of Formula I as shown in Scheme IXX.Therein tripeptide acylsulfonamide intermediate (1) is coupled to a1-chloro-3-bromoisoquinoline (2) using the previously described processof alkoxide displacement of an heteroarylhalo moiety to provideintermediate (3). The coupling of (1) and (2) is most efficient in thepresence of a catalyst such as lanthanum chloride as described herein.The isoquinoline ring system of intermediate (3) can be furtherfunctionalized by employing either Suzuki couplings (Process 1:subjecting (3) to heteroaryl or aryl boronic acids in the presence of apalladium catalyst such as palladium tetra(triphenylphosphine) and abase such as cesium carbonate in solvents such as DMF) or Stillecouplings (Process 2: subjecting (3) to heteraryl or aryl tinderivatives in the presence of palladium catalyst such as palladiumtetra(triphenylphosphine in solvents such as toluene).

Palladium reactions can also be employed to couple C4-amino prolineelements with functionalized heterocycles. Scheme XX shows intermediate(1) coupling with a functionalized isoquinoline i n the presence of apalladium catalyst and a base in a solvent such as toluene.Intermediates like (3) can be converted to compounds of Formula I usingthe methods described herein.

The construction of functionalized isoquinoline ring systems is alsopossible employing [4+2] cycloaddition reactions. For example (SchemeXXI) the use of vinyl isocyanates (1) in cycloaddition reactions withbenzyne precursors (2) provides functionalized isoquinolones (3). Saidisoquinolines can be incorporated into compounds of Formula I using themethods described herein.

Compounds of the invention can also be prepared by utilizing methodsknown to those skilled in the art, such as, for example, the methodsdescribed in patent application WO 03/099274, published Dec. 3, 2003,and WO 03/099316, published Dec. 4, 2003.

The present invention also provides compositions comprising a compoundof the present invention, or a pharmaceutically acceptable enantiomer,diastereomer, salt, solvate or prodrug thereof, and a pharmaceuticallyacceptable carrier. Pharmaceutical compositions of the present inventioncomprise a therapeutically effective amount of a compound of theinvention, or a pharmaceutically acceptable enantiomer, diastereomer,salt, solvate or prodrug thereof, and a pharmaceutically acceptablecarrier, with a pharmaceutically acceptable carrier, e.g., excipient, orvehicle diluent.

The active ingredient, i.e., compound, in such compositions typicallycomprises from 0.1 weight percent to 99.9 percent by weight of thecomposition, and often comprises from about 5 to 95 weight percent.

Thus, in one aspect of the invention, there is provided a compositioncomprising the compound of formula I and a pharmaceutically acceptablecarrier. Preferably, the composition further comprises a compound havinganti-HCV activity. As used herein, the term “anti-HCV activity” meansthe compound is effective to inhibit the function of a target selectedfrom the group consisting of HCV metalloprotease, HCV serine protease,HCV polymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly,HCV egress, HCV NS5A protein, IMPDH and a nucleoside analog for thetreatment of an HCV infection. Often, the other compound having anti-HCVactivity is effective to inhibit the function of target in the HCV lifecycle other than the HCV NS3 protease protein.

In one preferred aspect, the compound having anti-HCV activity is aninterferon. Preferably, the interferon is selected from the groupconsisting of interferon alpha 2B, pegylated interferon alpha, consensusinterferon, interferon alpha 2A, lymphoblastiod interferon tau.

In another aspect of the invention, the compound having anti-HCVactivity is selected from the group consisting of interleukin 2,interleukin 6, interleukin 12, a compound that enhances the developmentof a type 1 helper T cell response, interfering RNA, anti-sense RNA,Imiqimod, ribavirin, an inosine 5′-monophospate dehydrogenase inhibitor,amantadine, and rimantadine.

In one preferred aspect of the invention, the composition comprises acompound of the invention, an interferon and ribavirin.

In another preferred aspect of the invention, the compound havinganti-HCV activity is a small molecule compound. As used herein, the term“small molecule compound” means a compound having a molecular weight ofless than 1,500 daltons, preferably less than 1000 daltons. Preferably,the small molecule compound is effective to inhibit the function of atarget selected from the group consisting of HCV metalloprotease, HCVserine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCVentry, HCV assembly, HCV egress, HCV NS5A protein, inosine monophophatedehydrogenase (“IMPDH”) and a nucleoside analog for the treatment of anHCV infection.

Certain illustrative HCV inhibitor compounds which can be administeredwith the compounds of the present invention include those disclosed inthe following publications: WO 02/04425 A2 published Jan. 17, 2002, WO03/007945 A1 published Jan. 30, 2003, WO 03/010141 A2 published Feb. 6,2003, WO 03/010142 A2 published Feb. 6, 2003, WO 03/010143 A1 publishedFeb. 6, 2003, WO 03/000254 A1 published Jan. 3, 2003, WO 01/32153 A2published May 10, 2001, WO 00/06529 published Feb. 10, 2000, WO 00/18231published Apr. 6, 2000, WO 00/10573 published Mar. 2, 2000, WO 00/13708published Mar. 16, 2000, WO 01/85172 A1 published Nov. 15, 2001, WO03/037893 A1 published May 8, 2003, WO 03/037894 A1 published May 8,2003, WO 03/037895 A1 published May 8, 2003, WO 02/100851 A2 publishedDec. 19, 2002, WO 02/100846 A1 published Dec. 19, 2002, EP 1256628 A2published Nov. 13, 2002, WO 99/01582 published Jan. 14, 1999, WO00/09543 published Feb. 24, 2000.

Table 1 below lists some illustrative examples of compounds that can beadministered with the compounds of this invention. The compounds of theinvention can be administered with other anti-HCV activity compounds incombination therapy, either jointly or separately, or by combining thecompounds into a composition. TABLE 1 Type of Inhibitor or Brand NameTarget Source Company Omega IFN IFN-ω BioMedicines Inc., Emeryville, CABILN-2061 serine protease inhibitor Boehringer Ingelheim Pharma KG,Ingelheim, Germany Summetrel antiviral Endo Pharmaceuticals HoldingsInc., Chadds Ford, PA Roferon A IFN-α2a F. Hoffmann-La Roche LTD, Basel,Switzerland Pegasys PEGylated IFN-α2a F. Hoffmann-La Roche LTD, Basel,Switzerland Pegasys and PEGylated IFN- F. Hoffmann-La Roche Ribavirinα2a/ribavirin LTD, Basel, Switzerland CellCept HCV IgG F. Hoffmann-LaRoche immunosuppressant LTD, Basel, Switzerland Wellferon lymphoblastoidIFN- GlaxoSmithKline plc, αn1 Uxbridge, UK Albuferon - α albumin IFN-α2bHuman Genome Sciences Inc., Rockville, MD Levovirin ribavirin ICNPharmaceuticals, Costa Mesa, CA IDN-6556 caspase inhibitor IdunPharmaceuticals Inc., San Diego, CA IP-501 antifibrotic IndevusPharmaceuticals Inc., Lexington, MA Actimmune INF-γ InterMune Inc.,Brisbane, CA Infergen A IFN alfacon-1 InterMune Pharmaceuticals Inc.,Brisbane, CA ISIS 14803 antisense ISIS Pharmaceuticals Inc, Carlsbad,CA/Elan Phamaceuticals Inc., New York, NY JTK-003 RdRp inhibitor JapanTobacco Inc., Tokyo, Japan Pegasys and PEGylated IFN-α2a/ MaximPharmaceuticals Ceplene immune modulator Inc., San Diego, CA Cepleneimmune modulator Maxim Pharmaceuticals Inc., San Diego, CA Civacir HCVIgG Nabi immunosuppressant Biopharmaceuticals Inc., Boca Raton, FLIntron A and IFN-α2b/α1-thymosin RegeneRx Zadaxin BiopharmiceuticalsInc., Bethesda, MD/ SciClone Pharmaceuticals Inc, San Mateo, CALevovirin IMPDH inhibitor Ribapharm Inc., Costa Mesa, CA ViramidineIMPDH inhibitor Ribapharm Inc., Costa Mesa, CA Heptazyme ribozymeRibozyme Pharmaceuticals Inc., Boulder, CO Intron A IFN-α2bSchering-Plough Corporation, Kenilworth, NJ PEG-Intron PEGylated IFN-α2bSchering-Plough Corporation, Kenilworth, NJ Rebetron IFN-α2b/ribavirinSchering-Plough Corporation, Kenilworth, NJ Ribavirin ribavirinSchering-Plough Corporation, Kenilworth, NJ PEG-Intron/ PEGylated IFN-Schering-Plough Ribavirin α2b/ribavirin Corporation, Kenilworth, NJZadazim immune modulator SciClone Pharmaceuticals Inc., San Mateo, CARebif IFN-β1a Serono, Geneva, Switzerland IFN-β and EMZ701 IFN-β andEMZ701 Transition Therapeutics Inc., Ontario, Canada T67 β-tubulininhibitor Tularik Inc., South San Francisco, CA VX-497 IMPDH inhibitorVertex Pharmaceuticals Inc., Cambridge, MA VX-950/LY-570310 serineprotease inhibitor Vertex Pharmaceuticals Inc., Cambridge, MA/ Eli Lillyand Co. Inc., Indianapolis, IN Omniferon natural IFN-α Viragen Inc.,Plantation, FL XTL-002 monoclonal antibody XTL Biopharmaceuticals Ltd.,Rehovot, Isreal

The pharmaceutical compositions of this invention may be administeredorally, parenterally or via an implanted reservoir. Oral administrationor administration by injection are preferred. In some cases, the pH ofthe formulation may be adjusted with pharmaceutically acceptable acids,bases or buffers to enhance the stability of the formulated compound orits delivery form. The term parenteral as used herein includessubcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intrasynovial, intrasternal, intrathecal, andintralesional injection or infusion techniques.

When orally administered, the pharmaceutical compositions of thisinvention may be administered in any orally acceptable dosage formincluding, but not limited to, capsules, tablets, and aqueoussuspensions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions are administered orally, theactive ingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening and/or flavoring and/or coloring agents maybe added. Other suitable carriers for the above noted compositions canbe found in standard pharmaceutical texts, e.g. in “Remington'sPharmaceutical Sciences”, 19th ed., Mack Publishing Company, Easton,Pa., 1995.

The pharmaceutical compositions can be prepared by known proceduresusing well-known and readily available ingredients. The compositions ofthis invention may be formulated so as to provide quick, sustained ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art. In making thecompositions of the present invention, the active ingredient willusually be admixed with a carrier, or diluted by a carrier, or enclosedwithin a carrier which may be in the form of a capsule, sachet, paper orother container. When the carrier serves as a diluent, it may be asolid, semi-solid or liquid material which acts as a vehicle, excipientor medium for the active ingredient. Thus, the compositions can be inthe form of tablets, pills, powders, beadlets, lozenges, sachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as asolid or in a liquid medium), soft and hard gelatin capsules,suppositories, sterile injectable solutions, sterile packaged powdersand the like. Further details concerning the design and preparation ofsuitable delivery forms of the pharmaceutical compositions of theinvention are known to those skilled in the art.

Dosage levels of between about 0.01 and about 1000 milligram perkilogram (“mg/kg”) body weight per day, preferably between about 0.5 andabout 250 mg/kg body weight per day of the compounds of the inventionare typical in a monotherapy for the prevention and treatment of HCVmediated disease. Typically, the pharmaceutical compositions of thisinvention will be administered from about 1 to about 5 times per day oralternatively, as a continuous infusion. Such administration can be usedas a chronic or acute therapy. The amount of active ingredient that maybe combined with the carrier materials to produce a single dosage formwill vary depending upon the host treated and the particular mode ofadministration.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of theinfection, the patient's disposition to the infection and the judgmentof the treating physician. Generally, treatment is initiated with smalldosages substantially less than the optimum dose of the peptide.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. In general, thecompound is most desirably administered at a concentration level thatwill generally afford antivirally effective results without causing anyharmful or deleterious side effects.

When the compositions of this invention comprise a combination of acompound of the invention and one or more additional therapeutic orprophylactic agent, both the compound and the additional agent areusually present at dosage levels of between about 10 to 100%, and morepreferably between about 10 and 80% of the dosage normally administeredin a monotherapy regimen.

When these compounds or their pharmaceutically acceptable enantiomers,diastereomers, salts, solvates or prodrugs are formulated together witha pharmaceutically acceptable carrier, the resulting composition may beadministered in vivo to mammals, such as man, to inhibit HCV NS3protease or to treat or prevent HCV virus infection.

Accordingly, another aspect of this invention provides methods ofinhibiting HCV NS3 protease activity in patients by administering acompound of the present invention or a pharmaceutically acceptableenantiomer, diastereomer, salt or solvate thereof.

In one aspect of the invention, there is provided a method of treatingan HCV infection in a patient, comprising administering to the patient atherapeutically effective amount of the compound of the invention, or apharmaceutically acceptable enantiomer, diastereomer, solvate, prodrugor salt thereof.

Preferably, the method of administering the compound is effective toinhibit the function of the HCV NS3 protease protein. In a preferredaspect, the method further comprises administering another compoundhaving anti-HCV activity (as described above) prior to, after orconcurrently with a compound of the invention.

The compounds of the invention may also be used as laboratory reagents.Compounds may be instrumental in providing research tools for designingof viral replication assays, validation of animal assay systems andstructural biology studies to further enhance knowledge of the HCVdisease mechanisms. Further, the compounds of the present invention areuseful in establishing or determining the binding site of otherantiviral compounds, for example, by competitive inhibition.

The compounds of this invention may also be used to treat or preventviral contamination of materials and therefore reduce the risk of viralinfection of laboratory or medical personnel or patients who come incontact with such materials, e.g., blood, tissue, surgical instrumentsand garments, laboratory instruments and garments, and blood collectionor transfusion apparatuses and materials.

Further, the compounds and compositions of the invention can be used forthe manufacture of a medicament for treating HCV infection in a patient.

EXAMPLES

The specific examples that follow illustrate the syntheses of thecompounds of the instant invention, and are not to be construed aslimiting the invention in sphere or scope. The methods may be adapted tovariations in order to produce compounds embraced by this invention butnot specifically disclosed. Further, variations of the methods toproduce the same compounds in somewhat different manner will also beevident to one skilled in the art.

Solution percentages express a weight to volume relationship, andsolution ratios express a volume to volume relationship, unless statedotherwise. Nuclear magnetic resonance (NMR) spectra were recorded eitheron a Bruker 300, 400 or 500 MHz spectrometer; the chemical shifts (δ)are reported in parts per million. Flash chromatography was carried outon silica gel (SiO₂) according to Still's flash chromatography technique(W. C. Still et al., J. Org. Chem., (1978), 43, 2923).

All Liquid Chromatography (LC) data were recorded on a Shimadzu LC-10ASliquid chromatograph using a SPD-10AV UV-Vis detector and MassSpectrometry (MS) data were determined with a Micromass Platform for LCin electrospray mode (ES+).

Unless otherwise noted, in the following examples each compound wasanalyzed by LC/MS, using one of seven methodologies, having thefollowing conditions.

-   Columns: (Method A)—YMC ODS S7 C18 3.0×50 mm    -   (Method B)—YMC ODS-A S7 C18 3.0×50 mm    -   (Method C)—YMC S7 C18 3.0×50 mm    -   (Method D)—YMC Xterra ODS S7 3.0×50 mm    -   (Method E)—YMC Xterra ODS S7 3.0×50 mm    -   (Method F)—YMC ODS-A S7 C18 3.0×50 mm    -   (Method G)—YMC C18 S5 4.6×50 mm-   Gradient: 100% Solvent A/0% Solvent B to 0% Solvent A/100% Solvent B-   Gradient time: 2 min. (A, B, D, F, G); 8 min. (C, E)-   Hold time: 1 min. (A, B, D, F, G); 2 min. (C, E)-   Flow rate: 5 mL/min-   Detector Wavelength: 220 nm-   Solvent A: 10% MeOH/90% H₂O/0.1% TFA-   Solvent B: 10% H₂O/90% MeOH/0.1% TFA.

The abbreviations used in the present application, includingparticularly in the illustrative examples which follow, are well-knownto those skilled in the art. Some of the abbreviations used are asfollows: rt room temperature Boc tert-butyloxycarbonyl DMSOdimethylsulfoxide EtOAc ethyl acetate t-BuOK potassium t-butoxide Et₂Odiethyl ether TBME tert-butylmethyl ether THF tetrahydrofuran CDIcarbonyldiimidazole DBU 1,8-diazabicyclo[5.4.0]undec-7-ene TFAtrifluoroacetic acid NMM N-methylmorpholine HATUO-7-azabenzotriazol-1-yl HBTU O-{1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HOBT N-hydroxybenzotriazolePyBrop bromo-bis-pyrrolidine-phosphonium hexafluorophosphate DMFdimethylformamide MeOH methanol EDTA ethylenediaminetetraacetic acidHRMS high resolution mass spectrometry DMAP 4-dimethylaminopyridineDIPEA diisopropylethylamine DCM dichloromethane DCE dichloroethane

The compounds and chemical intermediates of the present invention,described in the following examples, were prepared according to thefollowing methods. It should be noted that the following exemplificationsection is presented in sections. Example numbers and compound numbersare not contiguous throughout the entire Examples portion of theapplication and hence, each section indicates a “break” in thenumbering. The numbering within each section is generally contiguous.

Section A:

Preparation of Intermediates:

Preparation of P1 Intermediates:

The PI intermediates described in this section can be used to preparecompounds of Formula I by the methods described herein.

I P1 elements:

1. Preparation of Racemic (1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropaneCarboxylic Acid Ethyl Ester

Method A Step 1

Glycine ethyl ester hydrochloride (303.8 g, 2.16 mole) was suspended intert-butylmethyl ether (1.6 L). Benzaldehyde (231 g, 2.16 mole) andanhydrous sodium sulfate (154.6 g, 1.09 mole) were added and the mixturecooled to 0° C. using an ice-water bath. Triethylamine (455 mL, 3.26mole) was added dropwise over 30 min and the mixture stirred for 48 h atrt. The reaction was then quenched by addition of ice-cold water (1 L)and the organic layer was separated. The aqueous phase was extractedwith tert-butylmethyl ether (0.5 L) and the combined organic phaseswashed with a mixture of saturated aqueous NaHCO₃ (1 L) and brine (1 L).The solution was dried over MgSO₄, concentrated in vacuo to afford 392.4g of the N-benzyl imine product as a thick yellow oil that was useddirectly in the next step. ¹H NMR (CDCl₃, 300 MHz) δ 1.32 (t, J=7.1 Hz,3H), 4.24 (q, J=7.1 Hz, 2H), 4.41 (d, J=1.1 Hz, 2H), 7.39-7.47 (m, 3H),7.78-7.81 (m, 2H), 8.31 (s, 1H).

Step 2

To a suspension of lithium tert-butoxide (84.06 g, 1.05 mol) in drytoluene (1.2 L), was added dropwise a mixture of the N-benzyl imine ofglycine ethyl ester (100.4 g, 0.526 mol) and trans-1,4-dibromo-2-butene(107.0 g, 0.500 mol) in dry toluene (0.6 L) over 60 min. Aftercompletion of the addition, the deep red mixture was quenched byaddition of water (1 L) and tert-butylmethyl ether (TBME, 1 L). Theaqueous phase was separated and extracted a second time with TBME (1 L).The organic phases were combined, 1 N HCl (1 L) was added and themixture stirred at room temperature for 2 h. The organic phase wasseparated and extracted with water (0.8 L). The aqueous phases were thencombined, saturated with salt (700 g), TBME (1 L) was added and themixture cooled to 0° C. The stirred mixture was then basified to pH 14by the dropwise addition of 10 N NaOH, the organic layer separated, andthe aqueous phase extracted with TBME (2×500 mL). The combined organicextracts were dried (MgSO₄) and concentrated to a volume of 1 L. To thissolution of free amine, was added BOC₂O or di-tert-butyldicarbonate(131.0 g, 0.6 mol) and the mixture stirred 4 days at rt. Additionaldi-tert-butyldicarbonate (50 g, 0.23 mol) was added to the reaction, themixture refluxed for 3 h, and was then allowed cool to room temperatureovernight. The reaction mixture was dried over MgSO₄ and concentrated invacuo to afford 80 g of crude material. This residue was purified byflash chromatography (2.5 Kg of SiO₂, eluted with 1% to 2% MeOH/CH₂Cl₂)to afford 57 g (53%) of racemicN-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid ethylester as a yellow oil which solidified while sitting in therefrigerator: ¹H NMR (CDCl₃, 300 MHz) δ 1.26 (t, J=7.1 Hz, 3H), 1.46 (s,9H), 1.43-1.49 (m, 1H), 1.76-1.82 (br m, 1H), 2.14 (q, J=8.6 Hz, 1H),4.18 (q, J=7.2 Hz, 2H), 5.12 (dd, J=10.3, 1.7 Hz, 1H), 5.25 (br s, 1H),5.29 (dd, J=17.6, 1.7 Hz, 1H), 5.77 (ddd, J=17.6, 10.3, 8.9 Hz, 1H); MSm/z 254.16 (M−1)Step 3 Preparation of Racemic (1R,2S)/(1S,2R)1-amino-2-vinylcyclopropane Carboxylic Acid Ethyl Ester Hydrochloride

N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid ethylester (9.39 g, 36.8 mmol) was dissolved in 4 N HCl/dioxane (90 ml, 360mmol) and was stirred for 2 h at rt. The reaction mixture wasconcentrated to supply (1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropanecarboxylic acid ethyl ester hydrochloride in quantitative yield (7 g,100%). ¹H NMR (methanol-d₄) δ 1.32 (t, J=7.1, 3H), 1.72 (dd, J=10.2, 6.6Hz, 1H), 1.81 (dd, J=8.3, 6.6 Hz, 1H), 2.38 (q, J=8.3 Hz, 1H), 4.26-4.34(m, 2H), 5.24 (dd, 10.3, 1.3 Hz, 1H), 5.40 (d, J=17.2, 1H), 5.69-5.81(m, 1H).Alternate Route for the Preparation of RacemicN-Boc-1-amino-2-vinylcyclopropane Carboxylic Acid Ethyl EsterHydrochloride

To a solution of potassium tert-butoxide (11.55 g, 102.9 mmol) in THF(450 mL) at −78° C. was added the commercially available N,N-dibenzylimine of glycine ethyl ester (25.0 g, 93.53 mmol) in THF (112 mL). Thereaction mixture was warmed to 0° C., stirred for 40 min, and was thencooled back to −78° C. To this solution was addedtrans-1,4-dibromo-2-butene (20.0 g, 93.50 mmol), the mixture stirred for1 h at 0° C. and was cooled back to −78° C. Potassium tert-butoxide(11.55 g, 102.9 mmol) was added, the mixture immediately warmed to 0°C., and was stirred one more hour before concentrating in vacuo. Thecrude product was taken up in Et₂O (530 mL), 1N aq. HCl solution (106mL, 106 mmol) added and the resulting biphasic mixture stirred for 3.5 hat rt. The layers were separated and the aqueous layer was washed withEt₂O (2×) and basified with a saturated aq. NaHCO₃ solution. The desiredamine was extracted with Et₂O (3×) and the combined organic extract waswashed with brine, dried (MgSO₄), and concentrated in vacuo to obtainthe free amine. This material was treated with a 4N HCl solution indioxane (100 mL, 400 mmol) and concentrated to afford(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid ethyl esterhydrochloride as a brown semisolid (5.3 g, 34% yield) identical to thematerial obtained from procedure A, except for the presence of a smallunidentified aromatic impurity (8%).Resolution of N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropaneCarboxylic Acid Ethyl Ester

Resolution A

To an aqueous solution of sodium phosphate buffer (0.1 M, 4.25 liter(“L”), pH 8) housed in a 12 Liter jacked reactor, maintained at 39° C.,and stirred at 300 rpm was added 511 grams of Acalase 2.4 L (about 425mL) (Novozymes North America Inc.). When the temperature of the mixturereached 39° C., the pH was adjusted to 8.0 by the addition of a 50% NaOHin water. A solution of the racemicN-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylic acid ethylester (85 g) in 850 mL of DMSO was then added over a period of 40 min.The reaction temperature was then maintained at 40° C. for 24.5 h duringwhich time the pH of the mixture was adjusted to 8.0 at the 1.5 h and19.5 h time points using 50% NaOH in water. After 24.5 h, theenantio-excess of the ester was determined to be 97.2%, and the reactionwas cooled to room temperature (26° C.) and stirred overnight (16 h)after which the enantio-excess of the ester was determined to be 100%.The pH of the reaction mixture was then adjusted to 8.5 with 50% NaOHand the resulting mixture was extracted with MTBE (2×2 L). The combinedMTBE extract was then washed with 5% NaHCO₃ (3×100 mL), water (3×100mL), and evaporated in vacuo to give the enantiomerically pureN-Boc-(1R,2S)/-1-amino-2-vinylcyclopropane carboxylic acid ethyl esteras light yellow solid (42.55 g; purity: 97% @ 210 nm, containing noacid; 100% enantiomeric excess (“ee”).

The aqueous layer from the extraction process was then acidified to pH 2with 50% H₂SO₄ and extracted with MTBE (2×2 L). The MTBE extract waswashed with water (3×100 mL) and evaporated to give the acid as lightyellow solid (42.74 g; purity: 99% @ 210 nm, containing no ester).

1R, 2S-ester

1S, 2R-acid ester acid High (+) ESI, C13H22NO4, [M+H]⁺, (−) ESI,C11H16NO4, Resolution cal. 256.1549, found 256.1542 [M−H]⁻, cal.226.1079, MassSpec found 226.1089 NMR observed chemical shift Solvent:CDCl₃ (proton δ 7.24 ppm, C-13 δ 77.0 ppm) Bruker DRX-500C: proton500.032 MHz, carbon 125.746 MHz Proton (pattern) C-13 Proton (pattern)C-13 Position ppm ppm ppm ppm 1 — 40.9 — 40.7 2 2.10 (q, J = 9.0 Hz)34.1 2.17 (q, J = 9.0 Hz) 35.0 3a 1.76 (br) 23.2 1.79 (br) 23.4 3b 1.46(br) 1.51, (br) 4 — 170.8 — 175.8 5 5.74 (ddd, J = 9.0, 133.7 5.75 (m)133.4 10.0, 17.0 Hz) 6a 5.25 (d, J = 17.0 Hz) 117.6 5.28 (d, J = 17.0Hz) 118.1 6b 5.08 (dd, J = 10.0, 1.5 5.12 (d, J = 10.5 Hz) Hz) 7 — 155.8— 156.2 8 — 80.0 — 80.6 9 1.43 (s) 28.3 1.43 (s) 28.3 10 4.16 (m) 61.3 —— 11 1.23 (t, J = 7.5 Hz) 14.2 — —Resolution B

To 0.5 mL 100 mM Heps.Na buffer (pH 8.5) in a well of a 24 well plate(capacity: 10 ml/well), 0.1 mL of Savinase 16.0 L (protease fromBacillus clausii) (Novozymes North America Inc.) and a solution of theracemic N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylicacid ethyl ester (10 mg) in 0.1 mL of DMSO were added. The plate wassealed and incubated at 250 rpm at 40° C. After 18 h, enantio-excess ofthe ester was determined to be 44.3% as following: 0.1 mL of thereaction mixture was removed and mixed well with 1 mL ethanol; aftercentrifugation, 10 microliter (“μl”) of the supernatant was analyzedwith the chiral HPLC. To the remaining reaction mixture, 0.1 mL of DMSOwas added, and the plate was incubated for additional 3 days at 250 rpmat 40° C., after which four mL of ethanol was added to the well. Aftercentrifugation, 10 μl of the supernatant was analyzed with the chiralHPLC and enantio-excess of the ester was determined to be 100%.

Resolution C

To 0.5 ml 100 mM Heps.Na buffer (pH 8.5) in a well of a 24 well plate(capacity: 10 mL/well), 0.1 ml of Esperase 8.0 L, (protease fromBacillus halodurans) (Novozymes North America Inc.) and a solution ofthe racemic N-Boc-(1R,2S)/(1S,2R)-1-amino-2-vinylcyclopropane carboxylicacid ethyl ester (10 mg) in 0.1 mL of DMSO were added. The plate wassealed and incubated at 250 rpm at 40° C. After 18 hour, enantio-excessof the ester was determined to be 39.6% as following: 0.1 mL of thereaction mixture was removed and mixed well with 1 mL ethanol; aftercentrifugation, 10 μl of the supernatant was analyzed with the chiralHPLC. To the remaining reaction mixture, 0.1 mL of DMSO was added, andthe plate was incubated for additional 3 days at 250 rpm at 40° C.,after which four mL of ethanol was added to the well. Aftercentrifugation, 10 μl of the supernatant was analyzed with the chiralHPLC and enantio-excess of the ester was determined to be 100%.

Samples analysis was carried out in the following manner:

1) Sample Preparation:

About 0.5 ml of the reaction mixture was mixed well with 10 volume ofEtOH. After centrifugation, 10 μl of the supernatant was injected ontoHPLC column.

2) Conversion Determination:

-   -   Column: YMC ODS A, 4.6×50 mm, S-5 μm    -   Solvent: A, 1 mM HCl in water; B, MeCN    -   Gradient: 30% B for 1 min; 30% to 45% B over 0.5 min; 45% B for        1.5 min; 45% to 30% B over 0.5 min.    -   Flow rate: 2 ml/min    -   UV Detection: 210 nm    -   Retention time: acid, 1.2 min; ester, 2.8 min.

3) Enantio-Excess Determination for the Ester:

-   -   Column: CHIRACEL OD-RH, 4.6×150 mm, S-5 μm    -   Mobile phase: MeCN/50 mM HClO₄ in water (67/33)    -   Flow rate: 0.75 ml/min.    -   UV Detection: 210 nm.

Retention Time:

-   -   (1S, 2R) isomer as acid: 5.2 min;    -   Racemate: 18.5 min and 20.0 min;    -   (1R, 2S) isomer as ester: 18.5 min.        2. Preparation of        N-Boc-(1R,2S)-1-amino-2-cyclopropylcyclopropane Carboxylic Acid        Ethyl Ester

A solution of N-Boc-(1R,2S)-1-amino-2-vinylcyclopropane carboxylic acid(255 mg, 1.0 mmol) in ether (10 mL) was treated with palladium acetate(5 mg, 0.022 mmol). The orange/red solution was placed under anatmosphere of N₂. An excess of diazomethane in ether was added dropwiseover the course of 1 h. The resulting solution was stirred at rt for 18h. The excess diazomethane was removed using a stream of nitrogen. Theresulting solution was concentrated by rotary evaporation to give thecrude product. Flash chromatography (10% EtOAc/hexane) provided 210 mg(78%) of N-Boc-(1R,2S)-1-amino-2-cyclopropylcyclopropane carboxylic acidethyl ester as a colorless oil. LC-MS (retention time: 2.13, similar tomethod A except: gradient time 3 min, Xterra MS C18 S7 3.0×50 mmcolumn), MS m/e 270 (M⁺+1).3. 1-tert-butoxycarbonylamino-cyclopropane-carboxylic Acid isCommercially Available

4. Preparation of 1-aminocyclobutanecarboxylic Acid MethylEster-hydrochloride

1-aminocyclobutanecarboxylic acid (100 mg, 0.869 mmol) (Tocris) wasdissolved in 10 mL of MeOH, HCl gas was bubbled in for 2 h. The reactionmixture was stirred for 18 h, and then concentrated in vacuo to give 144mg of a yellow oil. Trituration with 10 mL of ether provided 100 mg ofthe titled product as a white solid. ¹H NMR (CDCl₃) δ 2.10-2.25 (m, 1H),2.28-2.42 (m, 1H), 2.64-2.82 (m, 4H), 3.87 (s, 3H), 9.21 (br s, 3H).5. Preparation of Racemic (1R,2R)/(1S,2S)1-Amino-2-ethylcyclopropanecarboxylic Acid Tert-butyl Ester, ShownBelow.

Step 1: Preparation of 2-Ethylcyclopropane-1,1-dicarboxylic AcidDi-tert-butyl Ester, Shown Below.

To a suspension of benzyltriethylamrnonium chloride (21.0 g, 92.2 mmol)in a 50% aqueous NaOH solution (92.4 g in 185 mL H₂O) was added1,2-dibromobutane (30.0 g, 138.9 mmol) and di-tert-butylmalonate (20.0g, 92.5 mmol). The reaction mixture was vigorously stirred 18 h at rt, amixture of ice and water was then added. The crude product was extractedwith CH₂Cl₂ (3×) and sequentially washed with water (3×), brine and theorganic extracts combined. The organic layer was dried (MgSO₄), filteredand concentrated in vacuo. The resulting residue was flashchromatographed (100 g SiO₂, 3% Et₂O in hexane) to afford the titledproduct (18.3 g, 67.8 mmol, 73% yield) which was used directly in thenext reaction.Step 2: Preparation of Racemic 2-Ethylcyclopropane-1,1-dicarboxylic AcidTert-butyl Ester, Shown Below.

The product of Step 1 (18.3 g, 67.8 mmol) was added to a suspension ofpotassium tert-butoxide (33.55 g, 299.0 mmol) in dry ether (500 mL) at0° C., followed by H₂O (1.35 mL, 75.0 mmol) and was vigorously stirredovernight at rt. The reaction mixture was poured in a mixture of ice andwater and washed with ether (3×). The aqueous layer was acidified with a10% aq. citric acid solution at 0° C. and extracted with EtOAc (3×). Thecombined organic layers were washed with water (2×), brine, dried(MgSO₄) and concentrated in vacuo to afford the titled product as a paleyellow oil (10 g, 46.8 mmol, 69% yield).Step 3: Preparation of (1R,2R)/(1S,2S)2-Ethyl-1-(2-trimethylsilanylethoxycarbonylamino)cyclopropane-carboxylicAcid Tert-butyl Ester, Shown Below.

To a suspension, of the product of Step 2 (10 g, 46.8 mmol) and 3 g offreshly activated 4A molecular sieves in dry benzene (160 mL), was addedEt₃N (7.50 mL, 53.8 mmol) and DPPA (11 mL, 10.21 mmol). The reactionmixture was refluxed for 3.5 h, 2-trimethylsilyl-ethanol (13.5 mL, 94.2mmol) was then added, and the reaction mixture was refluxed overnite.The reaction mixture was filtered, diluted with Et₂O, washed with a 10%aqueous citric acid solution, water, saturated aqueous NaHCO₃, water(2×), brine (2×), dried (MgSO₄) and concentrated in vacuo. The residuewas suspended with 10 g of Aldrich polyisocyanate scavenger resin in 120mL of CH₂Cl₂, stirred at rt overnite and filtered to afford the titledproduct (8 g, 24.3 mmol; 52%) as a pale yellow oil: ¹H NMR (CDCl₃) δ0.03 (s, 9H), 0.97 (m, 5H), 1.20 (bm, 1H), 1.45 (s, 9H), 1.40-1.70 (m,4H), 4.16 (m, 2H), 5.30 (bs, 1H).Step 4: Preparation of Racemic (1R,2R)/(1S,2S)1-Amino-2-ethylcyclopropanecarboxylic Acid Tert-butyl Ester, ShownBelow.

To the product of Step 3 (3 g, 9 mmol) was added a 1.0 M TBAF solutionin THF (9.3 mL, 9.3 mmol) and the mixture heated to reflux for 1.5 h,cooled to rt and then diluted with 500 ml of EtOAc. The solution wassuccessively washed with water (2×100 mL), brine (2×100 mL), dried(MgSO₄), concentrated in vacuo to provide the title intermediate6.Preparation of 1-Amino-spiro[2.3]hexane-1-carboxylic Acid Methyl EsterHydrochloride Salt

Step 1 Preparation of [2,3]hexane-1,1-dicarboxylic Acid Dimethyl Ester,Shown Below.

To a mixture of methylene-cyclobutane (1.5 g, 22 mmol) and Rh₂(OAc)₄(125 mg, 0.27 mmol) in anhydrous CH₂Cl₂ (15 mL) was added 3.2 g (20mmol) of dimethyl diazomalonate (prepared according to J. Lee et al.Synth. Comm., 1995, 25, 1511-1515) at 0° C. over a period of 6 h. Thereaction mixture was then warmed to rt and stirred for another 2 h. Themixture was concentrated and purified by flash chromatography (elutingwith 10:1 hexane/Et₂O to 5:1 hexane/Et₂O) to give 3.2 g (72%) of[2,3]hexane-1,1-dicarboxylic acid dimethyl ester as a yellow oil. ¹H NMR(300 MHz, CDCl₃) δ 3.78 (s, 6 H), 2.36 (m, 2 H), 2.09 (m, 3 H), 1.90 (m,1 H), 1.67 (s, 2 H). LC-MS: MS m/z 199 (M⁺+1).Step 2: Preparation of Spiro[2,3]hexane-1,1-dicarboxylic Acid MethylEster, Shown Below.

To the mixture of spiro [2,3]hexane-1,1-dicarboxylic acid dimethyl ester(200 mg, 1.0 mmol) in 2 mL of MeOH and 0.5 mL of water was added KOH (78mg, 1.4 mmol). This solution was stirred at rt for 2 days. It was thenacidified with dilute HCl and extracted two times with ether. Thecombined organic phases were dried (MgSO₄) and concentrated to yield 135mg (73%) of 2 as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 3.78 (s, 3 H),2.36-1.90 (m, 8 H). LC-MS: MS m/z 185 (M⁺+1)

Step 3: Preparation of the Titled Product,1-amino-spiro[2.3]hexane-1-carboxylic Acid Methyl Ester HydrochlorideSalt.

To a mixture of spiro[2,3]hexane-1,1-dicarboxylic acid methyl ester (660mg, 3.58 mmol) in 3 mL of anhydrous t-BuOH was added 1.08 g (3.92 mmol)of DPPA and 440 mg (4.35 mmol) of Et₃N. The mixture was heated at refluxfor 21 h and then partitioned between H₂O and ether. The ether phase wasdried over magnesium sulfate, filtered and concentrated in vacuo toyield an oil. To this oil was added 3 mL of a 4 M HCl/dioxane solution.This acidic solution was stirred at rt for 2 h and then concentrated invacuo. The residue was triturated with ether to give 400 mg (58%) ofdesired product as a white solid. ¹H NMR (300 MHz, d6-DMSO) δ 8.96 (brs, 3 H), 3.71 (s, 3 H), 2.41 (m, 1 H), 2.12 (m, 4 H), 1.93 (m, 1 H),1.56 (q, 2 H, J=8 Hz). LC-MS of free amine: MS m/z 156 (M++1).7. Preparation of 1-Amino-spiro[2.4]heptane-1-carboxylic Acid MethylEster Hydrochloride Salt, Shown Below, was Prepared as Follows.

Step 1: Spiro[2.4]heptane-1,1-dicarboxylic Acid Dimethyl Ester, ShownBelow, was Prepared as Follows.

Using the same procedure described in the preparation of1-Amino-spiro[2.3]hexane-1-carboxylic acid methyl ester hydrochloridesalt 1.14 g (13.9 mmol) of methylenecyclopentane and 2.0 g (12.6 mmol)of dimethyl diazomalonate were reacted to yield 1.8 g (67%) of thedimethyl ester. ¹H NMR (300 MHz, CDCl₃) δ 3.73 (s, 6 H), 1.80 (m, 2 H),1.70 (m, 4 H), 1.60 (m, 4 H). LC-MS: MS m/z 213 (M⁺+1).Step 2: Preparation of Spiro[2.4]heptane-1,1-dicarboxylic Acid MethylEster, Shown Below, was Prepared as Follows.

Using the same procedure described in the preparation of1-Amino-spiro[2.3]hexane-1-carboxylic acid methyl ester hydrochloridesalt 1.7 g (8.0 mmol) of the product of Step 1 and 493 mg (8.8 mmol) ofKOH gave 1.5 g (94%) of spiro[2.4]heptane-1,1-dicarboxylic acid methylester. ¹H NMR (300 MHz, CDCl₃) δ 3.80 (s, 3 H), 2.06 (d, 1 H, J=5 Hz),1.99 (d, 1 H, J=5 Hz), 1.80-1.66 (m, 8 H). LC-MS: MS m/z 199 (M⁺+1).Step 3: Preparation of 1-Amino-spiro[2.4]heptane-1-carboxylic AcidMethyl Ester Hydrochloride Salt, Shown Below, was Prepared as Follows.

Using the same procedure described above in preparation of1-Amino-spiro[2.3]hexane-1-carboxylic acid methyl ester hydrochloridesalt, 500 mg (2.5 mmol) of the product of Step 2, 705 mg (2.5 mmol) ofDPPA and 255 mg (2.5 mmol) of Et₃N gave 180 mg (35%) of thishydrochloride salt. ¹H NMR (300 MHz, d6-DMSO) δ 8.90 (br s, 3 H), 3.74(s, 3 H), 1.84 (m, 1 H), 1.69 (m, 4 H), 1.58 (m, 4 H), 1.46 (d, 1 H, J=6Hz). LC-MS of free amine: MS m/z 170 (M⁺+1).8. Preparation of 1-Amino-spiro[2.2]pentane-1-carboxylic Acid MethylEster Hydrochloride Salt, Shown Below, was Prepared as Follows.

Step 1: Spiro[2.2]pentane-1,1-dicarboxylic Acid Dimethyl Ester, ShownBelow, was Prepared as Follows.

To a mixture of methylenecyclopropane (1.0 g, 18.5 mmol) (preparedaccording to P. Binger U.S. Pat. No. 5,723,714) and Rh₂(OAc)₄ (82 mg,0.185 mmol) in anhydrous CH₂Cl₂ (10 mL), was added dimethyldiazomalonate (2.9 g, 18.3 mmol) at 0° C. At the top of the flask wasinstalled a cold finger, the temperature of which was kept at −10° C.The reaction mixture was warmed to rt and stirred for another 2 h. Themixture was concentrated in vacuo and purified by flash chromatography(eluting with 10:1 hexane/Et₂O to 5:1 hexane/Et₂O) to give 0.85 g (25%)of the dimethyl ester as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 3.73(s, 6 H), 1.92 (s, 2 H), 1.04 (d, 4 H, J=3 Hz).Step 2: Spiro[2.2]pentane- 1,1-dicarboxylic Acid Methyl Ester, ShownBelow, was Prepared as Follows.

Using the same procedure described above in preparation of1-Amino-spiro[2.3]hexane-1-carboxylic acid methyl ester hydrochloridesalt, 800 mg (4.3 mmol) of the product of step 1 and 240 mg (4.3 mmol)of KOH gave 600 mg (82%) of Spiro[2.2]pentane-1,1-dicarboxylic acidmethyl ester. ¹H NMR (300 MHz, CDCl₃) δ 3.82 (s, 6 H), 2.35 (d, 1 H, J=3Hz), 2.26 (d, 1 H, J=3 Hz), 1.20 (m, 1 H), 1.15 (m, 1 H), 1.11 (m, 1 H),1.05 (m, 1 H). LRMS: MS m/z 169 (M⁺−1) (Method D).Step 3: 1-Amino-spiro[2.2]pentane-1-carboxylic Acid Methyl EsterHydrochloride Salt, Shown Below, was Prepared as Follows.

Using the same procedure described above for the preparation of1-Amino-spiro[2.3]hexane-1-carboxylic acid methyl ester hydrochloridesalt, 400 mg (2.3 mmol) of the product of step 2, 700 mg (2.5 mmol) ofDPPA and 278 mg (2.7 mmol) of Et₃N gave 82 mg (20%) of the hydrochloridesalt. ¹H NMR (300 MHz, CDCl₃) δ 9.19 (brs, 3 H), 3.81 (s, 3 H), 2.16 (d,J=5.5 Hz, 1 H), 2.01 (d, J=5.5 Hz, 1 H), 1.49 (m, 1 H), 1.24 (m, 1 H),1.12 (m, 2 H). LRMS of free amine: MS m/z 142 (M⁺+1).9. Preparation of 5-Amino-spiro[2.3]hexane-5-carboxylic Acid EthylEster, Shown Below, was Prepared as Follows.

Spiro[2.3]hexan-4-one (500 mg, 5 mmol), which was prepared frombicyclopropylidene (A. Meijere et al. Org. Syn. 2000, 78, 142-151)according to A. Meijere et al. J. Org. Chem. 1988, 53, 152-161, wascombined with ammonium carbamate (1.17 g, 15 mmol) and potassium cyanide(812 mg, 12.5 mmol) in 50 mL of EtOH and 50 mL of water. The mixture washeated at 55° C. for 2 days. Then NaOH (7 g, 175 mmol) was added and thesolution was heated under reflux overnight. The mixture was then chilledto 0° C., acidified to pH 1 with concentrated HCl, and concentrated invacuo. EtOH was added to the crude amino acid mixture and thenconcentrated to dryness (5×) so as to remove residual water. The residuedissolved in 100 mL of EtOH was cooled to 0° C. It was then treated with1 mL of SOCl₂ and refluxed for 3 days. The solids were removed byfiltration, and the filtrate was concentrated in vacuo to give the crudeproduct. The crude product was partitioned between 3 N NaOH, NaCl andEtOAc. The organic phase was dried over potassium carbonate andconcentrated. The residue was purified using column chromatography on C18 silica gel (eluting with MeOH/H₂O) to yield 180 mg (21%) of 15 as anoil. ¹H NMR (300 MHz, CDCl₃) δ8.20 (br s, 2 H), 4.27 (s, 2 H), 2.80 (s,1 H), 2.54 (s, 1 H), 2.34 (m, 2 H), 1.31 (s, 3 H), 1.02 (s, 1 H), 0.66(m, 3 H). ¹³C NMR (300 MHz, CDCl₃) δ 170.2(s), 63.0(s), 62.8 (s), 26.1(s), 26.0 (s), 24.9 (s), 13.9 (s), 11.4 (s), 10.9 (s). LC-MS: MS m/z 170(M⁺+1).II Heterocycles to be Used as Starting Material in the Construction ofP2 Elements for Subsequent Incorporation into Compounds of Formula I.

Isoquinoline (1) and substituted analogues thereof, can be incorporatedinto P2 elements using the two methods outline above and described indetail herein. Said P2 elements (3) can then be converted into compoundsof Formula I using procedures analogous to those described herein forsimilar isoquinoline analogues.

Isoxazole and oxazole heterocycle (1) and analogues thereof can beprepared using know chemistry and incorporated into compounds of FormulaI using the chemistry described herein for similar isoxazolepyridineintermediates as shown in section B.

Section B

Preparation of Compounds

The following LC MS conditions were used in the synthesis of thecompounds in section B

LC-MS Condition:

Columns: (Method A)—Xterra S7 C18 3.0×50 mm

Example 1000 Preparation of Intermediate 1

Step 1:

To a solution of 3-methoxy cinnamic acid (11.04 g, 62 mmol) andtriethylamine (12.52 g, 124 mmol) in acetone (80 mL) was added ethylchloroformate (approximately 1.5 equivalents) dropwise at 0° C. Afterstirring at this temperature for 1 h, aqueous NaN₃ (6.40 g, 100 mmol in35 mL H₂O) was added dropwise and the reaction mixture was stirred for16 h at the ambient temperature. Water (100 mL) was added to the mixtureand the volatile was removed in vacuo. The resulting slurry wasextracted with toluene (3×50 mL) and the combined organic layers weredried over MgSO₄. This dried solution was added dropwise to a heatedsolution of diphenylmethane (50 mL) and tributylamine (30 mL) at 190° C.The toluene was distilled off as added. After complete addition, thereaction temperature was raised to 210° C. for 2 h. After cooling, theprecipitated product was collected by filtration, washed with hexane(2×50 mL), and dried to yield the desired product as a white solid (5.53g, 51%) (Nicolas Briet at el., Tetrahedron, 2002, 5761-5766).

LC-MS (retention time: 0.82 min, method B), MS m/z 176 (M⁺+H).

Step 2:

6-Methoxy-2H-isoquinolin-1-one (5.0 g, 28.4 mmol) in POCl₃ (10 mL) washeated to gentle reflux for 3 h the evaporated in vacuo (Nicolas Brietat el., Tetrahedron, 2002, 5761-5766). The residue was poured into icedwater (20 mL) and neutralized to pH 10 with 10 M NaOH. Extracted withCHCl₃. The organic layer was washed with brine, dried over MgSO₄,filtered, evaporated. The residue was purified by flash chromatography(1:1 hexane-EtOAc) to afford 4.41 g (80%) of the desired product as awhite solid.

¹H NMR (CD₃OD) δ 3.98 (s, 3H), 7.34-7.38 (m, 2 H), 7.69 (d, J=5.5 Hz,1H), 8.10 (d, J=6.0 Hz, 1H), 8.23 (d, J=9.5 Hz, 1H);

LC-MS (retention time: 1.42 min, method B), MS m/z 194 (M⁺+H).

Step 3:

To a solution of N-BOC-3-(R)-hydroxy-L-proline (892 mg, 3.89 mmol) inDMSO (40 mL) at the ambient temperature was added potassiumtert-butoxide (1.34 g, 12.0 mmol) in one portion. The formed suspensionwas stirred at this temperature for 30 min before being cooled to 10° C.1-chloro-6-methoxy-isoquinolin (785 mg, 4.05 mmol) was added as solid inone portion and the final mixture was stirred at the ambient temperaturefor 12 h. Quenched with iced 5% citric acid (aq), extracted with EtOAC(100 mL). The aqueous phase was extracted with EtOAC again. The combinedorganic layers were washed with 5% citric acid (aq) and brinerespectively, dried over MgSO₄, filtered. The filtrate was evaporated invacuo to dryness to yield 1.49 g (99%) of the desired product as anoff-white foam. This material was used in the next step reaction ascrude without further purification.

¹H NMR (CD₃OD) δ 1.42, 1.44 (rotamers, 9H), 2.38-2.43 (m, 1H), 2.66-2.72(m, 1H), 3.80-3.87 (m, 2H), 3.92 (s, 3H), 4.44-4.52 (m, 1H), 5.73 (b,1H), 7.16-7.18 (m, 2H), 7.24-7.25 (m, 1H), 7.87-7.88 (m, 1H), 8.07 (d,J=8.5 Hz, 1H);

LC-MS (retention time: 1.62 min, method B), MS m/z 389 (M⁺+H).

Example 1001 Preparation of Compound 1001

Step 1:

A slurry of P2Boc-(4R)-(6-methoxy-isoquinoline-1-oxo)-S-proline]-P1(1R,2S VinylAcca)-COOEt (7.88 g, 14.99 mmol) in 4M HCl/dioxane (120 mL, 480 mmol)was stirred for 2 h, removed the solvent in vacuo and azeotroped withdry dioxane. To the residue was added DMF (75 mL), N-methylmorpholine(6.27 mL, 57.07 mmol), Boc-L-tert-leucine (5.20 g, 22.49 mmol), and HATU(8.53 g, 22.49 mmol). The reaction mixture was stirred at rt overniteand worked up by pouring the reaction mixture into ice water andadjusted to pH 5 with aqueous 1.0 N HCl and extracted with EtOAc. Theextract was washed with NaHCO₃ (aq.), brine, dried (MgSO₄) andconcentrated. The residue was purified over Biotage 65M column(EtOAc-hexanes: 5-100%) to provide the product (8.07 g, 84%): Retentiontime: 1.88 method C) MS m/z 639 (M⁺+1).

Step 2:

To a suspension of the product (4.0 g, 6.26 mmol) of Step 1 of Example384 {Boc-NH-P3(L-tert-BuGly)-P2[(4R)-(6-methoxyl-isoquinoline-1-oxo)-S-proline]-P1(1R,2SVinyl Acca)-COOEt} in THF(250 mL), CH₃OH (31 mL), and H₂O (125 mL) wasadded LiOH (2.4 g, 100.2 mmol). The reaction mixture was stirred forovernite and then adjusted to pH 7 with aqueous 1.0 N HCl. The organicsolvents were removed in vacuo. The aqueous residue was acidified to pH4 and extracted with EtOAc (2×). The combined organic solvent was dried(Na₂SO₄/MgSO₄), and concentrated in vacuo to supply the product (3.79 g,99%): ¹H NMR (methanol-d₄) δ ppm 1.05 (s, 9 H), 1.25 (m, 1 H), 1.29 (s,9 H), 1.46 (m, 1 H), 1.72 (dd, J=8.24, 5.19 Hz, 1 H), 2.23 (q, J=8.55Hz, 1 H), 2.68 (dd, J=13.89, 7.78 Hz, 1 H), 3.94 (s, 3 H), 4.05 (dd,J×11.60, 3.05 Hz, 1 H), 4.23 (d, J=8.85 Hz, 1 H), 4.46 (d, J=11.60 Hz, 1H), 4.63 (t, J=8.39 Hz, 1 H), 5.10 (d, J=10.38 Hz, 1 H), 5.29 (d,J=17.40 Hz, 1 H), 5.85 (m, 2 H), 7.10 (d, J=9.16 Hz, 1 H), 7.19 (s, 1H), 7.26 (d, J=5.49 Hz, 1 H), 7.91 (d, J=5.80 Hz, 1 H), 8.12 (d, J=9.16Hz, 1 H). LC-MS (Retention time: 1.81 method C) MS m/z 611 (M⁺+1).

Step 3:

A solution of CDI (0.143 g, 0.88 mmol) and the product (0.36 g, 0.59mmol) of Step 2 of Example 300{BOCNH-P3(L-t-BuGly)-P2[(4R)-6-methoxy-isoquinoline-1-oxo)-S-proline]-P1(1R,2SVinyl Acca)-CO₂H} in THF (8 mL) was heated at 70° C. for 60 min andallowed to cool down to rt. The reaction solution was evenly divided bysyringe into five flasks. To one of the flask was added4,5-dichloro-thiophene-2-sulfonic acid amide

(0.034 g, 0.15 mmol) and followed by the addition of a solution of neatDBU (0.022 mL, 0.15 mmol). The reaction was stirred for overnite, thenfiltered through syringe filter and purified by preparative HPLC(solvent B: 30% to 100%) and to provide the Compound 1001 (0.0335 mg);¹H NMR (500 MHz, Solvent: methanol-d₄) δ ppm 1.06 (s, 9 H), 1.30 (s, 9H), 1.40 (m, 1 H), 1.87 (dd, J=7.93, 5.49 Hz, 1 H), 2.23 (q, J=8.75 Hz,1 H), 2.31 (m, 1 H), 2.64 (dd, J=13.89, 7.17 Hz, 1 H), 3.95 (s, 3 H),4.08 (m, 1 H), 4.27 (s, 1 H), 4.47 (d, J=11.60 Hz, 1 H), 4.57 (dd,J=9.61, 7.78 Hz, 1 H), 5.05 (d, J=10.38 Hz, 1 H), 5.25 (d, J=17.09 Hz, 1H), 5.63 (s, 1 H), 5.85 (s, 1 H), 7.12 (d, J=8.85 Hz, 1 H), 7.21 (s, 1H), 7.27 (d, J=5.80 Hz, 1 H), 7.66 (s, 1 H), 7.91 (d, J=6.10 Hz, 1 H),8.12 (d, J=9.16 Hz, 1 H); LC-MS (Retention time: 2.04 method A), MS m/z824 (M⁺+1).

Example 1002 Preparation of Compound 1002

Compound 1002 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead of6-Ethoxy-benzothiazole-2-sulfonic acid amide (0.038 g, 0.15 mmol), wasused in the place of 4,5-dichloro-thiophene-2-sulfonic acid amide,purified by Prep-HPLC (solvent B: 30% to 100%) to provide the theCompound 1002 (0.0269 mg): ¹H NMR (300 MHz, Solvent methanol-d₄) δ ppm1.04 (s, 9 H), 1.28 (s, 9 H), 1.42 (m, 4 H), 1.77 (dd, J=8.05, 5.86 Hz,1 H), 2.20 (m, 1 H), 2.30 (m, 1 H), 2.62 (dd, J=13.54, 6.95 Hz, 1 H),3.92 (s, 3 H), 4.12 (m, 3 H), 4.26 (s, 1 H), 4.44 (d, J=10.98 Hz, 1 H),4.56 (dd, J=9.33, 7.50 Hz, 1 H), 4.79 (d, J=11.34 Hz, 1 H), 5.14 (d,J=17.57 Hz, 1 H), 5.40 (m, 1 H), 5.84 (s, 1 H), 7.10 (d, J=9.15 Hz, 1H), 7.23 (m, 3 H), 7.60 (d, J=2.20 Hz, 1 H), 7.89 (d, J=6.22 Hz, 1 H),7.96 (d, J=8.78 Hz, 1 H), 8.10 (d, J=9.15 Hz, 1 H); LC-MS (Retentiontime: 1.97 method A), MS m/z 851 (M⁺+1).

Example 1003 Preparation of Compound 1003

Compound 1003 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead of2,4-Dimethyl-thiazole-5-sulfonic acid amide (0.028 g, 0.15 mmol), wasused in the place of 4,5-dichloro-thiophene-2-sulfonic acid amide,purified by Prep-HPLC (solvent B: 30% to 100%) to provide the compound1003 (0.037 mg): 1H NMR (500 MHz, DMSO-D6) δ ppm 1.05 (s, 9 H), 1.28 (s,9 H), 1.36 (m, 1 H), 1.78 (d, J=6.41 Hz, 1 H), 2.18 (m, 1 H), 2.27 (m, 1H), 2.59 (s, 3 H), 2.61 (m, 1 H), 2.66 (s, 3 H), 3.92 (s, 3 H), 4.05 (d,J=9.16 Hz, 1 H), 4.26 (s, 1 H), 4.45 (d, J=11.90 Hz, 1 H), 4.55 (m, 1H), 4.95 (d, J=10.07 Hz, 1 H), 5.19 (d, J=17.09 Hz, 1 H), 5.40 (m, 1 H),5.82 (s, 1 H), 7.09 (d, J=8.85 Hz, 1 H), 7.17 (s, 1 H), 7.24 (d, J=5.49Hz, 1 H), 7.88 (d, J=5.80 Hz, 1 H), 8.09 (d, J=9.16 Hz, 1 H); LC-MS(Retention time: 1.84 method A), MS m/z 785 (M⁺+1).

Example 1004 Preparation of Compound 1004

Compound 1004 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead of5-Chloro-1,3-dimethyl-1H-pyrazole-4-sulfonic acid amide (0.031 g, 0.15mmol), was used in the place of 4,5-dichloro-thiophene-2-sulfonic acidamide, purified by Prep-HPLC (solvent B: 30% to 100%) to provide thecompound 1004 (0.0662 mg): ¹H NMR (500 MHz, Solvent methanol-d₄) δ ppm1.05 (s, 9 H), 1.28 (s, 9 H), 1.34 (dd, J=9.31, 5.65 Hz, 1 H), 1.75 (dd,J=7.93, 5.80 Hz, 1 H), 2.14 (m, 1 H), 2.28 (m, 1 H), 2.39 (s, 3 H), 2.61(dd, J=13.58, 6.87 Hz, 1 H), 3.78 (s, 3 H), 3.92 (s, 3 H), 4.06 (m, 1H), 4.26 (s, 1 H), 4.44 (d, J=10.99 Hz, 1 H), 4.56 (m, 1 H), 4.92 (dd,J=10.38, 1.53 Hz, 1 H), 5.17 (d, J=17.70 Hz, 1 H), 5.32 (m, 1 H), 5.83(s, 1 H), 7.09 (d, J=10.38 Hz, 1 H), 7.18 (s, 1 H), 7.25 (d, J=6.10 Hz,1 H), 7.89 (d, J=6.10 Hz, 1 H), 8.09 (d, J=9.16 Hz, 1 H); LC-MS(Retention time: 1.83 method A), MS m/z 802 (M⁺+1).

Example 1005 Preparation of Compound 1005

Compound 1005 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead of1-Methyl-1H-imidazole-4-sulfonic acid amide (0.022 g, 0.15 mmol), wasused in the place of 4,5-dichloro-thiophene-2-sulfonic acid amide,purified by Prep-HPLC (solvent B: 30% to 100%) to provide the compound1005 (0.0251 mg): ¹H NMR (300 MHz, Solvent methanol-d₄) δ ppm 1.02 (s, 9H), 1.28 (s, 9 H), 1.37 (dd, J=9.15, 5.49 Hz, 1 H), 1.75 (dd, J=7.87,5.67 Hz, 1 H), 2.13 (m, 1 H), 2.29 (m, 1 H), 2.61 (dd, J=13.72, 7.14 Hz,1 H), 3.78 (s, 3 H), 3.92 (s, 3 H), 4.06 (d, J=8.42 Hz, 1 H), 4.25 (s, 1H), 4.41 (d, J=12.08 Hz, 1 H), 4.53 (m, 1 H), 4.94 (d, J=10.25 Hz, 1 H),5.15 (d, J=17.20 Hz, 1 H), 5.42 (m, 1 H), 5.84 (s, 1 H), 7.09 (m, 1 H),7.18 (s, 1 H), 7.25 (d, J=5.86 Hz, 1 H), 7.70 (s, 1 H), 7.81 (s, 1 H),7.89 (d, J=5.86 Hz, 1 H), 8.09 (d, J=9.15 Hz, 1 H); LC-MS (Retentiontime: 1.69 method A), MS m/z 754 (M⁺+1).

Example 1006 Preparation of Compound 100620

Compound 1006 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead ofThiophene-2-sulfonic acid amide

(0.022 g, 0.13 mmol), was used in the place of4,5-dichloro-thiophene-2-sulfonic acid amide, purified by Prep-HPLC(solvent B: 30% to 100%) to provide the compound 1006 (0.0498 mg): ¹HNMR (500 MHz, BENZENE-D6) δ ppm 1.04 (s, 9 H), 1.27 (s, 9 H), 1.36 (m, 1H), 1.77 (m, 1 H), 2.17 (q, J-8.65 Hz, 1 H), 2.25 (m, 1 H) 2.59 (dd,J=13.58, 6.87 Hz, 1 H), 3.91 (s, 3 H), 4.04 (m, 1 H), 4.25 (s, 1 H),4.43 (d, J=11.60 Hz, 1 H), 4.53 (m, 1 H), 4.96 (d, J=10.38 Hz, 1 H),5.18 (d, J=17.40 Hz, 1 H), 5.50 (m, 1 H), 5.82 (s, 1 H), 7.11 (m, 2 H),7.17 (s, 1 H), 7.24 (d, J-5.49 Hz, 1 H), 7.80 (m, 1 H), 7.87 (dd,J=9.61, 5.65 Hz, 2 H), 8.08 (d, J=8.85 Hz, 1 H); LC-MS (Retention time:1.88 method A), MS m/z 756 (M⁺+1).

Example 1007 Preparation of Compound 1007

Compound 1007 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead ofBenzenesulfonamide (0.0179 g, 0.13 mmol), was used in the place of4,5-dichloro-thiophene-2-sulfonic acid amide, purified by Prep-HPLC(solvent B: 30% to 100%) to provide the compound 1007 (0.0507 mg): ¹HNMR (300 MHz, ACETONE-D6) δ ppm 1.05 (s, 9 H), 1.28 (s, 9 H), 1.34 (dd,J=9.33, 5.67 Hz, 1 H), 1.72 (dd, J=7.87, 5.67 Hz, 1 H), 2.22 (m, 2 H),2.61 (dd, J=13.17, 6.59 Hz, 1 H), 3.92 (s, 3 H), 4.06 (d, J=11.34 Hz, 1H), 4.27 (s, 1 H), 4.45 (d, J=11.71 Hz, 1 H), 4.55 (dd, J=9.88, 7.32 Hz,1 H), 5.17 (d, J=17.20 Hz, 1 H), 5.42 (m, 1 H), 5.83 (s, 1 H), 7.09 (m,1 H), 7.18 (s, 1 H), 7.25 (d, J=5.86 Hz, 1 H), 7.54 (m, 2 H), 7.66 (t,J=7.50 Hz, 1 H), 7.89 (d, J=5.86 Hz, 1 H), 7.97 (m, 2 H), 8.09 (d,J=9.15 Hz, 1 H)); LC-MS (Retention time: 1.89 method A), MS m/z 750(M⁺+1).

Example 1008 Preparation of Compound 1008

Compound 1008 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead of4-Methoxy-benzenesulfonamide (0.025 g, 0.13 mmol), was used in the placeof 4,5-dichoro-thiophene-2-sulfonic acid amide, purified by Prep-HPLC(solvent B: 30% to 100%) to provide the compound 1008 (0.004 mg): ¹H NMR(500 MHz, Solvent methanol-d₄) δ ppm 1.09 (s, 9 H), 1.31 (s, 9 H), 1.36(dd, J=9.31, 5.34 Hz, 1 H), 1.74 (dd, J=7.93, 5.80 Hz, 1 H), 2.17 (q,J=8.75 Hz, 1 H), 2.29 (m, 1 H), 2.63 (dd, J=13.73, 7.02 Hz, 1 H), 3.91(s, 3 H), 3.95 (s, 3 H), 4.09 (d, J=1 1.29 Hz, 1 H), 4.30 (s, 1 H), 4.47(d, J=11.90 Hz, 1 H), 4.56 (dd, J=9.92, 7.17 Hz, 1 H), 4.96 (d, J=10.38Hz, 1 H), 5.20 (d, J=17.09 Hz, 1 H), 5.48 (m, 1 H), 5.86 (s, 1 H), 7.07(m, 2 H), 7.12 (d, J=7.93 Hz, 1 H), 7.21 (s, 1 H), 7.28 (d, J=5.80 Hz, 1H), 7.93 (m, 3 H), 8.12 (d, J=8.85 Hz, 1 H); LC-MS (Retention time: 1.91method A), MS m/z 780 (M⁺+1).

Example 1009 Preparation of Compound 1009

Compound 1009 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead of4-Methyl-pyridine-2-sulfonic acid amide (0.023 g, 0.13 mmol), was usedin the place of 4,5-dichloro-thiophene-2-sulfonic acid amide, purifiedby Prep-HPLC (solvent B: 30% to 100%) to provide the compound 1009(0.0388 mg): ¹H NMR (500 MHz, Solvent methanol-d₄) δ ppm 1.06 (s, 9 H),1.31 (s, 9 H), 1.39 (m, 1 H), 1.75 (m, 1 H), 2.18 (m, 1 H), 2.34 (m, 1H), 2.46 (s, 3 H), 2.62 (m, 1 H), 3.95 (s, 3 H), 4.09 (m, 1 H), 4.28 (m,1 H), 4.45 (d, J=10.99 Hz, 1 H), 4.57 (m, 1 H), 4.93 (d, J=10.68 Hz, 1H), 5.19 (d, J=17.09 Hz, 1 H), 5.37 (m, 1 H), 5.86 (s, 1 H), 7.12 (d,J=8.55 Hz, 1 H), 7.21 (s, 1 H), 7.28 (d, J=5.80 Hz, 1 H), 7.92 (m, 2 H),8.01 (d, J=5.49 Hz, 1 H), 8.12 (d, J=8.85 Hz, 1 H), 8.49 (s, 1 H); LC-MS(Retention time: 1.85 method A), MS m/z 765 (M⁺+1).

Example 1010 Preparation of Compound 1010

Compound 1010 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead of6-Chloro-3-methyl-benzo[b]thiophene-2-sulfonic acid amide (0.035 g, 0.13mmol), was used in the place of 4,5-dichloro-thiophene-2-sulfonic acidamide, purified by Prep-HPLC (solvent B: 30% to 100%) to provide thecompound 1010 (0.0292 mg): ¹H NMR (300 MHz, Solvent methanol-d₄) δ ppm1.07 (s, 9 H), 1.29 (s, 9 H), 1.34 (m, 1 H), 1.74 (dd, J=7.87, 5.67 Hz,1 H), 2.16 (m, 1 H), 2.29 (m, 1 H), 2.62 (m, 1 H), 2.68 (s, 3 H), 3.92(s, 3 H), 4.06 (m, 1 H), 4.26 (s, 1 H), 4.46 (d, J=-12.08 Hz, 1 H), 4.59(m, 2 H), 5.10 (m, 1 H), 5.22 (m, 1 H), 5.82 (s, 1 H), 7.10 (d, J-9.15Hz, 1 H), 7.19 (d, J-2.20 Hz, 1 H), 7.25 (d, J=5.86 Hz, 1 H), 7.52 (dd,J=8.78, 1.83 Hz, 1 H), 7.91 (m, 3-H), 8.10 (d, J=8.78 Hz, 1 H); LC-MS(Retention time: 2.12 method A) MS m/z 854 (M⁺+1).

Example 1011 Preparation of Compound 1011

Compound 1011 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 insteadN-(5-Sulfamoyl-[1,3,4]thiadiazol-2-yl)-acetamide (0.0179 g, 0.13 mmol),was used in the place of 4,5-dichloro-thiophene-2-sulfonic acid amide,purified by Prep-HPLC (solvent B: 30% to 100%) to provide the compound1011 (0.0388 mg): ¹H NMR (500 MHz, Solvent methanol-d₄) δ ppm 1.03 (s, 9H), 1.28 (s, 9 H), 1.41 (m, 1 H), 1.81 (dd, J=8.24, 5.49 Hz, 1 H), 2.26(m, 2 H), 2.28 (s, 3 H), 2.61 (dd, J=14.04, 7.02 Hz, 1 H) 3.92 (s, 3 H),4.06 (m, 1 H), 4.24 (s, 1 H), 4.43 (d, J-1 1.60 Hz, 1 H), 4.55 (m, 1 H),4.99 (d, J=11.29 Hz, 1 H), 5.21 (d, J=17.09 Hz, 1 H), 5.52 (m, 1 H),5.83 (s, 1 H), 7.10 (d, J=8.85 Hz, 1 H), 7.18 (d, J=1.83 Hz, 1 H), 7.25(d, J=6.10 Hz, 1 H), 7.88 (d, J=5.80 Hz, 1 H), 8.09 (d, J=8.85 Hz, 1 H);LC-MS (Retention time: 1.84 method A), MS m/z 815 (M⁺+1).

Example 1012 Preparation of Compound 1012

Compound 1012 was prepared in the same procedure as described in Step 3of Example 1001 in preparation of Compound 1001 instead3,5-Dimethyl-isoxazole-4-sulfonic acid amide (0.033 g, 0.15 mmol), wasused in the place of 4,5-dichloro-5 thiophene-2-sulfonic acid amide,purified by Prep-HPLC (solvent B: 30% to 100%) to provide the compound1012 (0.0462 mg): ¹H NMR (500 MHz, Solvent methanol-d₄) δ ppm 1.08 (s, 9H), 1.30 (s, 9 H), 1.37 (dd, J-9.16, 5.49 Hz, 1 H), 1.80 (m, 1 H), 2.21(q, J=8.55 Hz, 1 H), 2.31 (m, 1 H), 2.40 (s, 3 H), 2.66 (m, 1 H), 2.68(s, 3 H), 3.95 (s, 3 H), 4.08 (d, J=8.85 Hz, 1 H), 4.28 (s, 1 H), 4.48(d, J=11.29 Hz, 1 H), 4.61 (m, 1 H), 4.98 (d, J=10.38 Hz, 1 H), 5.22 (d,J=17.09 Hz, 1 H), 5.36 (m, 1 H), 5.85 (s, 1 H), 7.12 (d, J=8.85 Hz, 1H), 7.21 (s, 1 H), 7.28 (d, J=6.10 Hz, 1 H), 7.91 (d, J=6.10 Hz, 1 H),8.13 (d, J=9.16 Hz, 1 H); LC-MS (Retention time: 2.05 method A), MS m/z769(M⁺+1).

Section C Biological Studies Recombinant HCV NS3/4A Protease ComplexFRET Peptide Assay

The purpose of this in vitro assay was to measure the inhibition of HCVNS3 protease complexes, derived from the BMS strain, H77 strain or J4L6Sstrain, as described below, by compounds of the present invention. Thisassay provides an indication of how effective compounds of the presentinvention would be in inhibiting HCV NS3 proteolytic activity.

Serum from an HCV-infected patient was obtained from Dr. T. Wright, SanFrancisco Hospital. An engineered full-length cDNA (complimentdeoxyribonucleic acid) template of the HCV genome (BMS strain) wasconstructed from DNA fragments obtained by reverse transcription-PCR(RT-PCR) of serum RNA (ribonucleic acid) and using primers selected onthe basis of homology between other genotype 1a strains. From thedetermination of the entire genome sequence, a genotype 1a was assignedto the HCV isolate according to the classification of Simmonds et al.(See P Simmonds, K A Rose, S Graham, S W Chan, F McOmish, B C Dow, E AFollett, P L Yap and H Marsden, J. Clin. Microbiol., 31(6), 1493-1503(1993)). The amino acid sequence of the nonstructural region, NS2-5B,was shown to be >97% identical to HCV genotype 1a (H77) and 87%identical to genotype 1b (J4L6S). The infectious clones, H77 (lagenotype) and J4L6S (1b genotype) were obtained from R. Purcell (NIH)and the sequences are published in Genbank (AAB67036, see Yanagi, M.,Purcell, R. H., Emerson, S. U. and Bukh, J. Proc. Natl. Acad. Sci.U.S.A. 94(16),8738-8743 (1997); AF054247, see Yanagi, M., St Claire, M.,Shapiro, M., Emerson, S. U., Purcell, R. H. and Bukh, J, Virology 244(1), 161-172. (1998)).

The H77 and J4L6S strains were used for production of recombinant NS3/4Aprotease complexes. DNA encoding the recombinant HCV NS3/4A proteasecomplex (amino acids 1027 to 1711) for these strains were manipulated asdescribed by P. Gallinari et al. (see Gallinari P, Paolini C, Brennan D,Nardi C, Steinkuhler C, De Francesco R. Biochemistry. 38(17):5620-32,(1999)). Briefly, a three-lysine solubilizing tail was added at the3′-end of the NS4A coding region. The cysteine in the P1 position of theNS4A-NS4B cleavage site (amino acid 1711) was changed to a glycine toavoid the proteolytic cleavage of the lysine tag. Furthermore, acysteine to serine mutation was introduced by PCR at amino acid position1454 to prevent the autolytic cleavage in the NS3 helicase domain. Thevariant DNA fragment was cloned in the pET21b bacterial expressionvector (Novagen) and the NS3/4A complex was expressed in Escherichia.coli strain BL21 (DE3) (Invitrogen) following the protocol described byP. Gallinari et al. (see Gallinari P, Brennan D, Nardi C, Brunetti M,Tomei L, Steinkuhler C, De Francesco R., J. Virol. 72(8):6758-69 (1998))with modifications. Briefly, the NS3/4A protease complex expression wasinduced with 0.5 millimolar (mM) Isopropyl β-D-1-thiogalactopyranoside(IPTG) for 22 hours (h) at 20° C. A typical fermentation (1 Liter (L))yielded approximately 10 grams (g) of wet cell paste. The cells wereresuspended in lysis buffer (10 mL/g) consisting of 25 mMN-(2-Hydroxyethyl)Piperazine-N′-(2-Ethane Sulfonic acid) (HEPES), pH7.5, 20% glycerol, 500 mM Sodium Chloride (NaCl), 0.5% Triton X-100, 1microgram/milliliter (“μg/mL”) lysozyme, 5 mM Magnesium Chloride(MgCl₂), 1 gg/ml DnaseI, 5mM β-Mercaptoethanol (βME), Proteaseinhibitor—Ethylenediamine Tetraacetic acid (EDTA) free (Roche),homogenized and incubated for 20 minutes (min) at 4° C. The homogenatewas sonicated and clarified by ultra-centrifugation at 235000 g for 1 hat 4° C. Imidazole was added to the supernatant to a final concentrationof 15 mM and the pH adjusted to 8.0. The crude protein extract wasloaded on a Nickel-Nitrilotriacetic acid (Ni-NTA) columnpre-equilibrated with buffer B (25 mM HEPES, pH 8.0, 20% glycerol, 500mM NaCl, 0.5% Triton X-100, 15 mM imidazole, 5 mM βME). The sample wasloaded at a flow rate of 1 mL/min. The column was washed with 15 columnvolumes of buffer C (same as buffer B except with 0.2% Triton X-100).The protein was eluted with 5 column volumes of buffer D (same as bufferC except with 200 mM Imidazole).

NS3/4A protease complex-containing fractions were pooled and loaded on adesalting column Superdex-S200 pre-equilibrated with buffer D (25mMHEPES, pH 7.5, 20% glycerol, 300 mM NaCl, 0.2% Triton X-100, 10 mM βME).Sample was loaded at a flow rate of 1 mL/min. NS3/4A proteasecomplex-containing fractions were pooled and concentrated toapproximately 0.5 mg/ml. The purity of the NS3/4A protease complexes,derived from the BMS, H77 and J4L6S strains, were judged to be greaterthan 90% by SDS-PAGE and mass spectrometry analyses.

The enzyme was stored at −80° C., thawed on ice and diluted prior to usein assay buffer. The substrate used for the NS3/4A protease assay wasRET S 1 (Resonance Energy Transfer Depsipeptide Substrate; AnaSpec, Inc.cat # 22991) (FRET peptide), described by Taliani et al. in Anal.Biochem. 240(2):60-67 (1996). The sequence of this peptide is looselybased on the NS4A/NS4B natural cleavage site for the HCV NS3 proteaseexcept there is an ester linkage rather than an amide bond at thecleavage site. The peptide substrate was incubated with one of the threerecombinant NS3/4A protease complexes, in the absence or presence of acompound of the present invention, and the formation of fluorescentreaction product was followed in real time using a Cytofluor Series4000.

The reagents were as follow: HEPES and Glycerol (Ultrapure) wereobtained from GIBCO-BRL. Dimethyl Sulfoxide (DMSO) was obtained fromSigma. β-Mercaptoethanol was obtained from Bio Rad.

Assay buffer: 50 mM HEPES, pH 7.5; 0.15 M NaCl; 0.1% Triton; 15%Glycerol; 10 mM βME. Substrate: 2 μM final concentration (from a 2 mMstock solution in DMSO stored at −20° C.). HCV NS3/4A protease type 1a(1b), 2-3 nM final concentration (from a 5 μM stock solution in 25 mMHEPES, pH 7.5, 20% glycerol, 300 mM NaCl, 0.2% Triton-X100, 10 mM βME).For compounds with potencies approaching the assay limit, the assay wasmade more sensitive by adding 50 μg/ml Bovine Serum Albumin (Sigma) tothe assay buffer and reducing the end protease concentration to 300 μM.

The assay was performed in a 96-well polystyrene black plate fromFalcon. Each well contained 25 μl NS3/4A protease complex in assaybuffer, 50 μl of a compound of the present invention in 10% DMSO/assaybuffer and 25 μl substrate in assay buffer. A control (no compound) wasalso prepared on the same assay plate. The enzyme complex was mixed withcompound or control solution for 1 min before initiating the enzymaticreaction by the addition of substrate. The assay plate was readimmediately using the Cytofluor Series 4000 (Perspective Biosystems).The instrument was set to read an emission of 340 nm and excitation of490 nm at 25° C. Reactions were generally followed for approximately 15min.

The percent inhibition was calculated with the following equation:100−[(δF _(inh) /δF _(con))×100]where δF is the change in fluorescence over the linear range of thecurve. A non-linear curve fit was applied to theinhibition-concentration data, and the 50% effective concentration(IC₅₀) was calculated by the use of Excel X1-fit software using theequation, y=A+((B−A)/(1+((C/x){circumflex over ( )}D))).

All of the compounds tested were found to inhibit the activity of theNS3/4A protease complex with IC50's of 1.2 μM or less. Further,compounds of the present invention, which were tested against more thanone type of NS3/4A complex, were found to have similar inhibitoryproperties though the compounds uniformly demonstrated greater potencyagainst the 1b strains as compared to the 1a strains.

Specificity Assays

The specificity assays were performed to demonstrate the in vitroselectivity of the compounds of the present invention in inhibiting HCVNS3/4A protease complex as compared to other serine or cysteineproteases.

The specificities of compounds of the present invention were determinedagainst a variety of serine proteases: human neutrophil elastase (HNE),porcine pancreatic elastase (PPE) and human pancreatic, chymotrypsin andone cysteine protease: human liver cathepsin B. In all cases a 96-wellplate format protocol using colorimetric p-nitroaniline (pNA) substratespecific for each enzyme was used as described previously (PCT PatentApplication No. WO 00/09543) with some modifications to the serineprotease assays. All enzymes were purchased from Sigma while thesubstrates were from Bachem.

Each assay included a 2 h enzyme-inhibitor pre-incubation at roomtemperature followed by addition of substrate and hydrolysis to −30%conversion as measured on a Spectramax Pro microplate reader. Compoundconcentrations varied from 100 to 0.4 μM depending on their potency.

The final conditions for each assay were as follows:

-   -   50 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)        pH 8, 0.5 M Sodium Sulfate (Na₂SO₄), 50 mM NaCl, 0.1 mM EDTA, 3%        DMSO, 0.01% Tween-20 with:    -   133 μM succ-AAA-pNA and 20 nM HNE or 8 nM PPE; 100 μM        succ-AAPF-pNA and 250 μM Chymotrypsin.    -   100 mM NaHPO₄ (Sodium Hydrogen Phosphate) pH 6, 0.1 mM EDTA, 3%        DMSO, 1 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride),        0.01% Tween-20, 30 μM Z-FR-pNA and 5 nM Cathepsin B (enzyme        stock activated in buffer containing 20 mM TCEP before use).

The percentage of inhibition was calculated using the formula:[1−((UV _(inh) −UV _(bank))/(UV _(ctl) −UV _(blank)))]×100

A non-linear curve fit was applied to the inhibition-concentration data,and the 50% effective concentration (IC₅₀) was calculated by the use ofExcel X1-fit software.

Generation of HCV Replicon

An HCV replicon whole cell system was established as described byLohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R.,Science 285(5424):110-3 (1999). This system enabled us to evaluate theeffects of our HCV Protease compounds on HCV RNA replication. Briefly,using the HCV strain 1b sequence described in the Lohmann paper(Assession number: AJ238799), an HCV cDNA was generated encoding the 5′internal ribosome entry site (IRES), the neomycin resistance gene, theEMCV (encephalomyocarditis virus)-IRES and the HCV nonstructuralproteins, NS3-NS5B, and 3′ non-translated region (NTR). In vitrotranscripts of the cDNA were transfected into the human hepatoma cellline, Huh7. Selection for cells constitutively expressing the HCVreplicon was achieved in the presence of the selectable marker, neomycin(G418). Resulting cell lines were characterized for positive andnegative strand RNA production and protein production over time.

FRET Assay

Huh7 cells, constitutively expressing the HCV replicon, were grown inDulbecco's Modified Eagle Media (DMEM) containing 10% Fetal calf serum(FCS) and 1 mg/ml G418 (Gibco-BRL). Cells were seeded the night before(1.5×10⁴ cells/well) in 96-well tissue-culture sterile plates. Compoundand no compound controls were prepared in DMEM containing 4% FCS, 1:100Penicillin/Streptomysin, 1:100 L-glutamine and 5% DMSO in the dilutionplate (0.5% DMSO final concentration in the assay). Compound/DMSO mixeswere added to the cells and incubated for 4 days at 37° C. After 4 days,plates were rinsed thoroughly with Phosphate-Buffered Saline (PBS) (3times 150 μl). The cells were lysed with 25 μl of a lysis assay reagentcontaining the FRET peptide (RET S1, as described for the in vitroenzyme assay). The lysis assay reagent was made from 5× cell Luciferasecell culture lysis reagent (Promega #E153A) diluted to 1× with distilledwater, NaCl added to 150 mM final, the FRET peptide diluted to 10 μMfinal from a 2 mM stock in 100% DMSO. The plate was then placed into theCytofluor 4000 instrument which had been set to 340 nm excitation/490 nmemission, automatic mode for 21 cycles and the plate read in a kineticmode. EC₅₀ determinations were carried out as described for the IC₅₀determinations.

Luciferase Assay

As a secondary assay, EC₅₀ determinations from the replicon FRET assaywere confirmed in a luciferase reporter assay. Utilization of a repliconluciferase reporter assay was first described by Krieger et al (KriegerN, Lohmann V, and Bartenschlager R, J. Virol. 75(10):4614-4624 (2001)).The replicon construct described for our FRET assay was modified byreplacing the resistance gene neomycin with the Blasticidin-resistancegene fused to the N-terminus of the humanized form of Renilla luciferase(restriction sites Asc1/Pme1 used for the subcloning). The adaptivemutation at position 1179 (serine to isoleucine) was also introduced(Blight K J, Kolykhalov, A A, Rice, C M, Science 290(5498): 1972-1974).

The luciferase reporter assay was set up by seeding huh7 cells the nightbefore at a density of 2×10⁶ cells per T75 flask. Cells were washed thenext day with 7.5 ml Opti-MEM. Following the Invitrogen protocol, 40 μlDMRIE-C was vortexed with 5 ml Opti-MEM before adding 5 μg HCV reporterreplicon RNA. The mix was added to the washed huh7 cells and left for 4h at 37° C. In the mean time, serial compound dilutions and no compoundcontrols were prepared in DMEM containing 10% FCS and 5% DMSO in thedilution plate (0.5% DMSO final concentration in the assay).

Compound/DMSO mixes were added to each well of a 24-well plate. After 4h, the transfection mix was aspirated, and cells washed with 5 ml ofOpti-MEM before trypsinization. Trypsinized cells were resuspended in10% DMEM and seeded at 2×10⁴ cells/well in the 24-well plates containingcompound or no compound controls. Plates were incubated for 4 days.After 4 days, media was removed and cells washed with PBS. 100 μl 1×Renilla Luciferase Lysis Buffer (Promega) was immediately added to eachwell and the plates either frozen at −80° C. for later analysis, orassayed after 15 min of lysis. Lysate (40 μl) from each well wastransferred to a 96-well black plate (clear bottom) followed by 200 μl1× Renilla Luciferase assay substrate. Plates were read immediately on aPackard TopCount NXT using a luminescence program.

The percentage inhibition was calculated using the formula below:% control=average luciferase signal in experimentalwells(+compound)/average luciferase signal in DMSO controlwells(−compound)The values were graphed and analyzed using XLFit to obtain the EC₅₀value.

Compounds in accordance with the present invention were tested forbiological activity as described above and found to have activities inthe ranges as follow:

-   IC50 Activity Ranges (NS3/4A BMS Strain): A is 10-100 micromolar    (μM); B is 1-10 μM; C is 0.1-1 μM; D is <0.1 μM-   EC50 Activity Range (for compounds tested): A is 10-100 μM; B is    1-10 μM; C is 0.1-1 μM; D is <0.1 μM    Note that by using the Patent example number and the Patent compound    number shown in Table 2, the structures of compounds can be found    herein.

In accordance with the present invention, preferably the compounds havea biological activity (EC₅₀) of 10 μM or less, more preferably 1 μM orless and most preferably 100 nM or less. TABLE 2 Biological activityExample & Compound Number IC50 (uuM) EC50 (uM) Example 1001 Compound1001 D B Ex. 1002, Cmpd. 1002 C B Ex. 1003, Cmpd. 1003 C C Ex. 1004,Cmpd. 1004 C C Ex. 1005, Cmpd. 1005 B B Ex. 1006, Cmpd. 1006 D C

Those skilled in the art will recognize that although the invention hasbeen described above with respect to specific aspects, other aspects areintended to be within the scope of the claims which follow. Alldocuments referenced herein are hereby incorporated by reference as ifset out in full.

1. A compound having the formula

(a) R₁ is Het or aryl; (b) m is 1 or 2; (c) n is 1 or 2; (d) R₂ is H; orC₁₋₆ alkyl, C₂₋₆ alkenyl or C₃₋₇ cycloalkyl, each optionally substitutedwith halogen; (e) R₃ is C₁₋₈ alkyl optionally substituted with halo,cyano, amino, C₁₋₆ dialkylamino, C₆₋₁₀ aryl, C₇₋₁₄ alkylaryl, C₁₋₆alkoxy, carboxy, hydroxy, aryloxy, C₇₋₁₄ alkylaryloxy, C₂₋₆ alkylesteror C₈₋₁₅ alkylarylester; C₃₋₁₂ alkenyl, C₃₋₇ cycloalkyl, or C₄₋₁₀alkylcycloalkyl, wherein the cycloalkyl or alkylcycloalkyl areoptionally substituted with hydroxy, C₁₋₆ alkyl, C₂₋₆ alkenyl or C₁₋₆alkoxy; or R₃ together with the carbon atom to which it is attachedforms a C₃₋₇ cycloalkyl group optionally substituted with C₂₋₆ alkenyl;(f) Y is H, phenyl substituted with nitro, pyridyl substituted withnitro, or C₁₋₆ alkyl optionally substituted with cyano, OH or C₃₋₇cycloalkyl; provided that if R₄ or R₅ is H then Y is H; (g) B is H, C₁₋₆alkyl, R₄—(C═O)—, R₄O(C═O)—, R₄—N(R₅)—C(═O)—, R₄—N(R₅)—C(═S)—, R₄SO₂—,or R₄—N(R₅)—SO₂—; (h) R₄ is (i) C₁₋₁₀ alkyl optionally substituted withphenyl, carboxyl, C₁₋₆ alkanoyl, 1-3 halogen, hydroxy, —OC(O)C₁₋₆ alkyl,C₁₋₆ alkoxy, amino optionally substituted with C₁₋₆ alkyl, amido, or(lower alkyl) amido; (ii) C₃₋₇ cycloalkyl, C₃₋₇ cycloalkoxy, or C₄₋₁₀alkylcycloalkyl, each optionally substituted with hydroxy, carboxyl,(C₁₋₆ alkoxy)carbonyl, amino optionally substituted with C₁₋₆ alkyl,amido, or (lower alkyl) amido; (iii) C₆₋₁₀ aryl or C₇₋₁₆ arylalkyl, eachoptionally substituted with C₁₋₆ alkyl, halogen, nitro, hydroxy, amido,(lower alkyl) amido, or amino optionally substituted with C₁₋₆ alkyl;(iv) Het; (v) bicyclo(1.1.1)pentane; or (vi) —C(O)OC₁₋₆ alkyl,C₂₋₆alkenyl or C₂₋₆ alkynyl; (i) R₅ is H; C₁₋₆ alkyl optionallysubstituted with 1-3 halogens; or C₁₋₆ alkoxy provided R₄ is C₁₋₁₀alkyl; (j) X is O, S, SO, SO₂, OCH₂, CH₂O or NH; (k) R′ is Het, C₆₋₁₀aryl or C₇₋₁₄ alkylaryl, each optionally substituted with R^(a); and (l)R^(a) is C₁₋₆alkyl, C₃₋₇ cycloalkyl, C₁₋₆ alkoxy, C₃₋₇ cycloalkoxy,halo-C₁₋₆ alkyl, CF₃, mono-or di-halo-C₁₋₆ alkoxy, cyano, halo,thioalkyl, hydroxy, alkanoyl, NO₂, SH, , amino, C₁₋₆ alkylamino, di(C₁₋₆) alkylamino, di (C₁₋₆) alkylamide, carboxyl, (C₁₋₆) carboxyester,C₁₋₆ alkylsulfone, C₁₋₆ alkylsulfonamide, di (C₁₋₆) alkyl(alkoxy)amine,C₆₋₁₀ aryl, C₇₋₁₄ alkylaryl, or a 5-7 membered monocyclic heterocycle;or a pharmaceutically acceptable enantiomer, diastereomer, salt, solvateor prodrug thereof.
 2. A compound of claim 1 wherein R₁ is


3. The compound of claim 1 wherein R₂ is C₁₋₆ alkyl, C₂₋₆ alkenyl orC₃₋₇ cycloalkyl.
 4. The compound of claim 3 wherein R₂ is C₂₋₆ alkenyl.5. The compound of claim 1 wherein R₃ is C₁₋₈ alkyl optionallysubstituted with C₆aryl, C₁₋₆ alkoxy, carboxy, hydroxy, aryloxy, C₇₋₁₄alkylaryloxy, C₂₋₆ alkylester or C₈₋₁₅ alkylarylester; C₃₋₁₂ alkenyl;C₃₋₇ cycloalkyl; or C₄₋₁₀ alkylcycloalkyl.
 6. The compound of claim 5wherein R₃ is C₁₋₈ alkyl optionally substituted with C₁₋₆ alkoxy; orC₃₋₇ cycloalkyl.
 7. The compound of claim 1 wherein Y is H.
 8. Thecompound of claim 1 wherein B is H, C₁₋₆ alkyl, R₄—(C═O)—, R₄O(C═O)—,R₄—N(R₅)—C(═O)—, R₄—N(R₅)—C(═S)—, R₄SO₂—, or R₄—N(R₅)—SO₂—.
 9. Thecompound of claim 8 wherein B is R₄—(C═O)—, R₄O(C═O)—, orR₄—N(R₅)—C(═O)—
 10. The compound of claim 9 wherein B is R₄O(C═O)— andR₄ is C₁₋₆alkyl.
 11. The compound of claim 1 wherein R₄ is (i) C₁₋₁₀alkyl optionally substituted with phenyl, carboxyl, C₁₋₆ alkanoyl, 1-3halogen, hydroxy, C₁₋₆ alkoxy; (ii) C₃₋₇ cycloalkyl, C₃₋₇ cycloalkoxy,or C₄₋₁₀ alkylcycloalklyl; or (iii) C₆₋₁₀ aryl or C₇₋₁₆ arylalkyl, eachoptionally substituted with C₁₋₆ alkyl or halogen.
 12. The compound ofclaim 11 wherein R₄ is (i) C₁₋₁₀ alkyl optionally substituted with 1-3halogen or C₁₋₆ alkoxy; or (ii) C₃₋₇ cycloalkyl or C₄₋₁₀alkylcycloalkyl.
 13. The compound of claim 1 wherein R₅ is H or C₁₋₆alkyl optionally substituted with 1-3 halogens.
 14. The compound ofclaim 13 wherein R₅ is H.
 15. The compound of claim 1 wherein X is O orNH.
 16. The compound of claim 1 wherein R′ is Het or C₆₋₁₀ aryl eachoptionally substituted with R^(a).
 17. The compound of claim 16 whereinR′ is Het.
 18. The compound of claim 17 wherein the heterocycle contains1 or 2 nitrogen atoms and optionally a sulfur atom or an oxygen atom inthe ring.
 19. The compound of claim 18 wherein the heterocycle issubstituted with at least one of C₁₋₆ alkyl, C₁₋₆ alkoxy, halo, C₆₋₁₀aryl, C₇₋₁₄ alkylaryl, or a 5-7 membered monocyclic heterocycle.
 20. Thecompound of claim 1 wherein R^(a) is C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₁₋₆alkoxy, halo-C₁₋₆ alkyl, halo, amino, C₆ aryl, or a 5-7 memberedmonocyclic heterocycle.
 21. A compound having the formula

wherein: (a) R₁ is unsubstituted Het or Het substituted with from one tothree of halo, cyano, trifluoromethyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, amido,C₁₋₆ amino, phenyl, said phenyl being unsubstituted or substituted withone to three substituents selected from halo, cyano, nitro, C₁₋₆ alkyl,C₁₋₆ alkoxy, amido or phenyl; or a monocyclic aryl; (b) R₂ is C₁₋₆alkyl, C₂₋₆ alkenyl or C₃₋₇ cycloalkyl; (c) R₃ is C₁₋₈ alkyl optionallysubstituted with C₆ aryl, C₁₋₆ alkoxy, carboxy, hydroxy, aryloxy, C₇₋₁₄alkylaryloxy, C₂₋₆ alkylester, C₈₋₁₅ alkylarylester; C₃₋₁₂ alkenyl, C₃₋₇cycloalkyl, or C₄₋₁₀ alkylcycloalkyl; (d) B is H, C₁₋₆ alkyl, R₄—(C═O)—,R₄O(C═O)—, R₄—N(R₅)—C(═O)—, R₄—N(R₅)—C(═S)—, R₄SO₂—, or R₄—N(R₅)—SO₂—;(e) R₄ is (i) C₁₋₁₀ alkyl optionally substituted with phenyl, carboxyl,C₁₋₆ alkanoyl, 1-3 halogen, hydroxy, C₁₋₆ alkoxy; (ii) C₃₋₇ cycloalkyl,C₃₋₇ cycloalkoxy, or C₄₋₁₀ alkylcycloalklyl; or (iii) C₆₋₁₀ aryl orC₇₋₁₆ arylalkyl, each optionally substituted with C₁₋₆ alkyl or halogen;(f) R₅ is H or C₁₋₆ alkyl optionally substituted with 1-3 halogens; (g)X is O or NH; (h) R′ is Het; or C₆₋₁₀ aryl optionally substituted withR^(a); and (i) R^(a) is C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₁₋₆ alkoxy,halo-C₁₋₆ alkyl, halo, amino, C₆ aryl, or a 5-7 membered monocyclicheterocycle; or a pharmaceutically acceptable enantiomer, diastereomer,salt, solvate or prodrug thereof.
 22. The compound of claim 21 whereinR′ is a bicyclic heterocycle.
 23. The compound of claim 22 wherein theheterocycle contains 1 or 2 nitrogen atoms and optionally a sulfur atomor an oxygen atom in the ring.
 24. The compound of claim 22 wherein theheterocycle is substituted with at least one of C₁₋₆ alkyl, C₁₋₆ alkoxy,halo, C₆ aryl, and a 5-7 membered monocyclic heterocycle.
 25. Thecompound of claim 21 wherein R′ is a bicyclic heterocycle containing 1nitrogen atom and substituted with methoxy and at least one of a C₆ aryland a 5-7 membered monocyclic heterocycle.
 26. The compound of claim 21wherein R′ is a monocyclic heterocycle.
 27. The compound of claim 26wherein the heterocycle contains 1 or 2 nitrogen atoms and optionally asulfur atom or an oxygen atom in the ring.
 28. The compound of claim 26wherein the heterocycle is substituted with at least one of C₁₋₆ alkyl,C₁₋₆ alkoxy, halo, C₆₋₁₀ aryl, C₇₋₁₄ alkylaryl, or a 5-7 memberedmonocyclic heterocycle.
 29. The compound of claim 21 wherein R′ is amonoyclic heterocycle containing 1 or 2 nitrogen atoms and substitutedwith methoxy and at least one of a C₆ aryl and a 5-7 membered monocyclicheterocycle.
 30. A compound having the formula

wherein: (a) R₁ is unsubstituted Het or Het substituted with one tothree of halo, cyano, trifluoromethyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, amido,or amino; or a C₆ aryl; (b) R₂ is C₂₋₆ alkenyl; (c) R₃ is C₁₋₈ alkyl;(d) B is R₄O(C═O)—, or R₄—N(H)—C(═O)—; (e) R₄ is C₁₋₁₀ alkyl; (f) R′ isa bicyclic heterocycle optionally substituted with R^(a); and (g) R^(a)is C₁₋₆ alkyl, C₁₋₆ alkoxy, halo, C₆ aryl, or a 5-7 membered monocyclicheterocycle; or a pharmaceutically acceptable enantiomer, diastereomer,salt, solvate or prodrug thereof.
 31. The compound of claim 30 whereinR₂ is vinyl.
 32. The compound of claim 30 wherein R₃ is t-butyl.
 33. Thecompound of claim 30 wherein R₄ is t-butyl.
 34. The compound of claim 30wherein R′ is quinoline or isoquinoline optionally substituted withR^(a).
 35. The compound of claim 30 wherein R₂ is vinyl, R₃ is t-butyl,R₄ is t-butyl, and R′ is isoquinoline substituted with R^(a).
 36. Thecompound of claim 35 wherein R^(a) is C₁₋₆ alkoxy.
 37. The compound ofclaim 36 wherein R^(a) further includes at least one of C₆ aryl or a 5-7membered monocyclic heterocycle.
 38. A composition comprising thecompound of claim I and a pharmaceutically acceptable carrier.
 39. Thecomposition according to claim 38 further comprising a compound havinganti-HCV activity.
 40. The composition according to claim 39 wherein thecompound having anti-HCV activity is an interferon.
 41. The compositionaccording to claim 40 wherein the interferon is selected from the groupconsisting of interferon alpha 2B, pegylated interferon alpha, consensusinterferon, interferon alpha 2A, and lymphoblastiod interferon tau. 42.The composition according to claim 39 wherein the compound havinganti-HCV activity is selected from the group consisting of interleukin2, interleukin 6, interleukin 12, a compound that enhances thedevelopment of a type I helper T cell response, interfering RNA,anti-sense RNA, Imiqimod, ribavirin, an inosine 5′-monophospatedehydrogenase inhibitor, amantadine, and rimantadine.
 43. Thecomposition according to the claim 38 further comprising an interferonand ribavirin.
 44. The composition according to claim 39 wherein thecompound having HCV activity is a small molecule compound.
 45. Thecomposition according to claim 39 wherein the compound having anti-HCVactivity is effective to inhibit the function of a target selected fromthe group consisting of HCV metalloprotease, HCV serine protease, HCVpolymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCVegress, HCV NS5A protein, IMPDH and a nucleoside analog for thetreatment of an HCV infection.
 46. A method of inhibiting the functionof the HCV serine protease comprising contacting the HCV serine proteasewith the compound of claim
 1. 47. A method of treating an HCV infectionin a patient, comprising administering to the patient a therapeuticallyeffective amount of the compound of claim 1, or a pharmaceuticallyacceptable enantiomer, diastereomer, solvate, prodrug or salt thereof.48. The method according to claim 47 wherein the compound is effectiveto inhibit the function of the HCV serine protease.
 49. The methodaccording to claim 47 further comprising administering another compoundhaving anti-HCV activity prior to, after or simultaneously with thecompound of claim
 1. 50. The method according to claim 49 wherein theother compound having anti-HCV activity is an interferon.
 51. The methodaccording to claim 50 wherein the interferon is selected from the groupconsisting of interferon alpha 2B, pegylated interferon alpha, consensusinterferon, interferon alpha 2A, lymphoblastiod interferon tau.
 52. Themethod according to claim 49 wherein the other compound having anti-HCVactivity is selected from the group consisting of interleukin 2,interleukin 6, interleukin 12, a compound that enhances the developmentof a type I helper T cell response, interfering RNA, anti-sense RNA,Imiqimod, ribavirin, an inosine 5′-monophospate dehydrogenase inhibitor,amantadine, and rimantadine.
 53. The method according to claim 52wherein the compound having anti-HCV activity is a small molecule. 54.The method according to claim 53 wherein the compound having anti-HCVactivity is effective to inhibit the function of a target selected fromthe group consisting of HCV metalloprotease, HCV serine protease, HCVpolymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCVegress, HCV NS5A protein, IMPDH and a nucleoside analog for thetreatment of an HCV infection.
 55. The method according to claim 49wherein the other compound having anti-HCV activity is effective toinhibit the function of target in the HCV life cycle other than the HCVserine protease.
 56. Use of the compound of claim 1 for the manufactureof a medicament for treating HCV infection in a patient.
 57. Use of thecomposition of claim 38 for the manufacture of a medicament for treatingHCV infection in a patient.